Plasmids for transforming plant cells

ABSTRACT

This invention relates to several plasmids which are useful for genetically transforming plant cells. A first plasmid, such as pMON120, contains a T-DNA border, one or more marker genes, a unique cleavage site, and a region of Ti plasmid homology. A gene which is expressed in plant cells may be inserted into this plasmid to obtain a derivative plasmid, such as pMON128 which expresses neomycin phosphotransferase in plant cells. The derivative plasmid is inserted into a suitable microorganism, such as  A. tumefaciens  which contains a Ti plasmid. The inserted plasmids recombine with Ti plasmids to form co-integrate plasmids. Only a single crossover event is required to create the desired co-integrate plasmid.  A. tumefaciens  cells with co-integrate plasmids are selected and co-cultured with plant cells. The co-integrate Ti plasmids enter the plant cells and insert a segment of T-DNA which does not contain tumorigenic genes into the plant genome. The transformed plant cell(s) may be regenerated into a morphologically normal plant which will pass the inserted gene(s) to its descendants.

This is a continuation of application Ser. No. 06/458,411, filed Jan.17, 1983, now abandoned, and a continuation-in-part of application Ser.No. 06/458,414, filed Jan. 17, 1983, now abandoned.

TECHNICAL FIELD

This invention is in fields of genetic engineering, plant biology, andbacteriology.

BACKGROUND ART

In the past decade, the science of genetic engineering has developedrapidly. A variety of processes are known for inserting a heterologousgene into bacteria, whereby the bacteria become capable of efficientexpression of the inserted genes. Such processes normally involve theuse of plasmids which may be cleaved at one or more selected cleavagesites by restriction endonucleases. Typically, a gene of interest isobtained by cleaving one piece of DNA, and the resulting DNA fragment ismixed with a fragment obtained by cleaving a vector such as a plasmid.The different strands of DNA are then connected (“ligated”) to eachother to form a reconstituted plasmid. See, for example, U.S. Pat. Nos.4,237,224 (Cohen and Boyer, 1980); 4,264,731 (Shine, 1981); 4,273,875(Manis, 1981); 4,322,499 (Baxter et al, 1982), and 4,336,336 (Silhavy etal, 1982).

A variety of other reference works are available. Some of these worksdescribe the natural process whereby DNA is transcribed into mRNA andmRNA is translated into protein, see, e.g., L. Stryer, Biochemistry, 2ndedition, p 559 et seq. (W. H. Freeman and Co., 1981); A. L. Lehninger,Biochemistry, 2nd edition, p. 853 et seq. (Worth Publ., 1975). Otherworks describe methods and products of genetic manipulation; see, e.g.,T. Maniatis et al, Molecular Cloning, A Laboratory Manual (Cold SpringHarbor Labs, 1982); J. K. Setlow and A. Hollaender, Genetic Engineering,Principles and Methods (Plenum Press, 1979). Hereafter, all referenceswill be cited in abbreviated form; a list of complete citations isincluded after the Examples.

Most of the genetic engineering work performed to date involves theinsertion of genes into various types of cells, primarily bacteria suchas E. coli, various other microorganisms such as yeast, and mammaliancells. However, many of the techniques and substances used for geneticengineering of animal cells and microorganisms are not directlyapplicable to genetic engineering involving plants.

As used herein, the term “plant” refers to a multicellulardifferentiated organism that is capable of photosynthesis, such asangiosperms and multicellular algae. This does not includemicroorganisms, such as bacteria, yeast, and fungi. The term “plantcell” includes any cell derived from a plant; this includesundifferentiated tissue such as callus or crown gall tumor, as well asplant seeds, propagules, pollen, or plant embryos.

Ti and Ri Plasmids

The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens has beenproposed for use as a natural vector for introducing foreign geneticinformation into plant cells (Hernalsteen et al 1980; Rorsch andSchilperoort, 1978). Certain types of A. tumefaciens are capable ofinfecting a wide variety of plant cells, causing crown gall disease. Theprocess of infection is not fully understood. At least part of the Tiplasmid enters the plant cell. Various metabolic alterations occur, andpart of the Ti plasmid is inserted into the genome of the plant(presumably into the chromosomes). The part of the Ti plasmid thatenters the plant genome is designated as “transferred DNA” (T-DNA).T-DNA is stably maintained in the plant DNA (Chilton et al, 1977; Yadevet al, 1980; Willmitzer et al, 1980; Otten et al, 1981).

Research by several laboratories has led to the characterization ofseveral structural (i.e., protein coding) genes located in T-DNA(Garfinkel et al 1981; Leemans et al 1982), as well as other DNAsequences which appear to serve various other functions. For example,two sequences referred to as the “left border” and the “right border”appear to delineate T-DNA and may be involved in the process wherebyT-DNA is transferred into plant chromosomes (Zambryski et al 1982).

A different species of Agrobacterium, A. rhizogenes, carries a“root-inducing” (Ri) plasmid which is similar to the Ti plasmid.Infection of a plant cell by A. rhizogenes causes hairy root disease.Like the Ti plasmid, a segment of DNA called “T-DNA” (also referred toby some researchers as “R-DNA”) is transferred into the plant genome ofan infected cell.

Various other bacteria are also reported to be capable of causinggenetic transformation of plant cells, including A. rubi and certainbacteria of the genus Rhizobia which have been treated with a mutagenicagent. Hooykaas et al, at page 156 of Setlow and Holaender, 1979.

As used herein, the term plant tumor inducing plasmid includes anyplasmid (1) which is contained in a microorganism, other than a virus,which is capable of causing genetic transformation of one or more typesof plants or plant cells, and (2) which contains a segment of DNA whichis inserted into a plant genome. This includes Ri plasmids.

As used herein, the ter “T-DNA” refers to a segment of DNA in or fromPlant tumor inducing plasmid (1) which has been inserted into the genomeof one or more types of plant cells, or (2) which is contained in asegment of DNA that is located between two sequences of bases which arecapable of serving as T-DNA borders. As used herein, the terms “T-DNAborder” and “border” are determined and applied empirically; these termsshall refer to a sequence of bases which appears at or near the end of asegment of DNA which is transferred from a plant tumor inducing plasmidinto a plant genome.

Despite the existing knowledge of T-DNA and plant tumor inducingplasmids, no one prior to this invention has been able to utilize thesevectors for the introduction of foreign genes which are expressed ingenetically modified plants. A variety of obstacles to such use havebeen encountered in genetic engineering efforts. Such obstacles include:

1) the large size (approximately 200,000 base pairs) and resultingcomplexity of plant tumor inducing plasmids preclude the use of standardrecombinant DNA techniques to genetically modify and/or insert foreigngenes into specific sites in the T-DNA. For example, there are no knownunique restriction endonuclease cleavage sites in a Ti plasmid (Leemanset al, 1982).

2) the T-DNA, which is inserted into and expressed in plant cells,contains genes which are involved in the production of high levels ofphytohormones in the transformed plant cells (Leemans et al 1982). Thehigh levels of phytohormones interfere with the normal metabolic andregenerative process of the cells, and prevent the formation ofphenotypically normal plants from the cells (Braun and Wood, 1976; Yanget al, 1980). Exceptions to this are rare cases where the T-DNA hasundergone extensive spontaneous deletions in planta to eliminate thosegenes involved in phytohormone production. Under these conditions,normal plants are reported to be obtainable at low frequency (Otten etal, 1981). However, the T-DNA genes involved in phytohormone productioncould not be deleted prior to this invention, since they were veryimportant in the identification and/or selection of transformed plantcells (Marton et al, 1979):

As described above, simple recombinant DNA techniques for introducingforeign genes into plasmids are not applicable to the large Ti plasmid.As a result, several indirect methods have been developed and arediscussed below. The first reported use of the Ti plasmid as a vectorwas in model experiments in which bacterial transposons were insertedinto T-DNA and subsequently introduced into plant cells. The bacterialtransposons were reported to be stably maintained in the plant genome(Hernalsteens et al, 1980; Garfinkel et al 1981). However, in thesecases the transformed tumor tissues were found to be incapable ofregeneration into normal plants, and there was no reported evidence forthe expression of bacterial genes in the plant cells. In addition,because the insertion of bacterial transposons is believed to beessentially random, a great deal, of effort was required to identify andlocalize the position of the inserted DNA in these examples. Therefore,this approach is not likely to be useful to introduce desired genes in apredictable manner into plants.

Other researchers have reported the use of intermediate vectors whichreplicate in both E. coli and A. tumefaciens (Matzke and Chilton, 1981;Leemans et al 1981; Garfinkel et al, 1981). The intermediate vectorscontain relatively small subfragments of the Ti plasmid which can bemanipulated using standard recombinant DNA techniques. The subfragmentscan be modified by the deletion of specific sequences or by theinsertion of foreign genes at specific sites. The intermediate vectorscontaining the modified T DNA subfragment are then introduced into A.tumefaciens by transformation or conjugation. Double recombinationbetween the modified T-DNA fragment on the intermediate vector and itswild-type counterpart on the Ti plasmid results in the replacement ofthe wild-type copy with the modified fragment. Cells which contain therecombined Ti plasmids can be selected using appropriate antibiotics.

Various foreign DNA's have been inserted at specific sites in the T-DNAby this method and they have been reported to be stably transferred intoplant cells (Matzke and Chilton, 1981, Leemans et al 1981, 1982).However, such foreign genes have not been reported to be capable o′ fexpression in plant cells, and the transformed plant cells remainincapable of regeneration into normal plants. Furthermore, in theprocedure described above, it is preferred for a double crossover eventto occur, in order to substitute the modified DNA fragment for thewild-type copy. A single crossover results in the formation of aco-integrate plasmid which contains two copies of the T-DNAsubfragments. This duplication is undesirable in these methods sincehomologous recombination, which can occur in A. tumefaciens cells or inplant cells, can result in the loss of the inserted foreign gene(s).

A major disadvantage of the above methods is that the frequency ofdouble recombination is quite low, about 10⁻⁴ to 10⁻⁹ (Leemans et al,1981) and it requires extensive effort to identify and isolate the raredouble-crossover recombinants. As a result, the number and types ofexperiments which can be performed using existing methods forgenetically engineering the Ti plasmid is severely limited.

Other Means for Inserting DNA into Plant Cells

A variety of other methods have been reported for inserting DNA intoplant cells. One such method involves the use of lipid vesicles, alsocalled liposomes, to encapsulate one or more DNA molecules. Theliposomes and their DNA contents may be taken up by plant cells; see,e.g., Lurquin, 1981. If the inserted DNA can be incorporated into theplant genome, replicated, and inherited, the plant cells will betransformed.

Other alternate techniques involve contacting plant cells with DNA whichis complexed with either (a) polycationic substances, such aspoly-L-ornithine (Davey et al, 1980), or (b) calcium phosphate (Krens etal, 1982). Using these techniques, all or part of a Ti plasmid has beenreportedly inserted into plant cells, causing tumorigenic alteration ofthe plant cells.

Another method has been developed involving the fusion of bacteria,which contain desired plasmids, with plant cells. Such methods involveconverting the bacteria into spheroplasts and converting the plant cellsinto protoplasts. Both of these methods remove the cell wall barrierfrom the bacterial and plant cells, using enzymic digestion. The twocell types can then be used together by exposure to chemical agents,such as polyethylene glycol. See Hasezawa et al, 1981.

However, all of the foregoing techniques suffer from one or more of thefollowing problems:

1. transformation efficiencies reported to date have been very low;

2. only small DNA molecules can be inserted into plant cells;

3. only small numbers of DNA molecules can be inserted into plant cells;and/or,

4. a gene which is inserted into a plant cell will not be stablymaintained by the plant cell unless it is incorporated into the genomeof the plant cell, i.e., unless the gene is inserted into a chromosomeor plasmid that replicates in the plant cell.

For these and possibly other reasons, no one has yet reported expressionof a gene inserted into a plant cell by any of the foregoing techniques,except for the tumorigenic transformations noted above.

Prior to this invention, no satisfactory method existed for the creationand identification of genetically transformed plant cells which could beroutinely regenerated into morphologically normal plants.

SUMMARY OF THE INVENTION

This invention relates to several plasmids which are useful for creatingtransformed plant cells which are capable of subsequent regenerationinto differentiated, morphologically-normal plants. This invention alsorelates to microorganisms containing such plasmids, and to methods forcreating such plasmids and microorganisms.

This invention involves a first plasmid, such as pMON120, which hascertain desired characteristics described below. A gene which is capableof being expressed in plant cells may be inserted into this plasmid toobtain a derivative plasmid, such as pMON128. For example, plasmidpMON128 contains a chimeric gene which expresses neomycinphosphotransferase II (NPT II), an enzyme which inactivates certainantibiotics. The chimeric gene is capable of expression in plant cells.

The derivative plasmid is inserted into a suitable microorganism, suchas Agrobacterium tumefaciens cells which contain plant tumor inducingplasmids. In the A. tumefaciens cells, some of the inserted plasmidsrecombine with, plant tumor inducing plasmids to form a co-integrateplasmid; this is due to a region of homology between the two plasmids.Only a single crossover event is required to create the desiredco-integrate plasmid.

Because of the characteristics of the inserted plasmid this invention,the resulting co-integrate plant tumor inducing contain the chimericgene and/or any other inserted gene within the T-DNA region of theco-integrate plasmid. The inserted gene(s) are surrounded by at leasttwo T-DNA borders, at least one of which was inserted into the planttumor inducing plasmid by the crossover event. By means of appropriateantibiotics, A. tumefaciens cells which do not have co-integrate planttumor inducing plasmids with inserted genes are killed.

A. tumefaciens cells with co-integrate plasmids are co-cultured withplant cells, such as protoplasts, protoplast-derived cells, plantcuttings, or intact plants, under conditions which allow theco-integrate plant tumor inducing plasmids, or portions thereof, toenter the plant cells. Once inside the plant cells, a portion of theplant tumor inducing plasmid which is surrounded by the two T-DNAborders is inserted by natural processes into the plant genome. Thissegment of DNA contains the chimeric gene and/or any other desiredgene(s). Preferably, the segment of vector DNA which is inserted intothe plant genome does not contain any genes which would render the plantcell incapable of being regenerated into a differentiated,morphologically-normal plant. The transformed plant cell(s) may beregenerated into a morphologically-normal plant which will pass theinserted gene to its descendants.

A variety of uses exist for plants transformed by the method of thisinvention. For example, a gene which codes for an enzyme, whichinactivates a herbicide may be inserted into a plant. Alternately, agene which codes for a desired mammalian polypeptide such as growthhormone, insulin, interferon, or somatostatin may be inserted intoplants. The plants may be grown and harvested, and the polypeptide couldbe extracted from the plant tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart indicating the steps of this invention, usingpMON128 and the NOS-NPT II gene as an example.

FIG. 2 represents the creation of pMON41, a plasmid used to constructpMON120.

FIG. 3 represents the creation of M-4, an M13-derived DNA used toconstruct pMON109.

FIG. 4 represents the creation of pMON54, a plasmid used to constructpMON109.

FIG. 5 represents the creation of pMON109, a plasmid, used to constructpMON120.

FIG. 6 represents the creation of pMON113, a plasmid used to constructpMON120.

FIG. 7 represents the creation of plasmid pMON120, an intermediatevector with three restriction endonuclease cleavage sites which aresuitable for the insertion of a desired gene.

FIG. 8 represents the creation of pMON128, an intermediate vector whichwas obtained by inserting a chimeric NOS-NPT II kanamycin-resistancegene into pMON120.

FIG. 9 represents the cointegration of pMON128 with a wild-type Tiplasmid by means of a single crossover event, thereby creating aco-integrate plasmid with multiple borders.

FIG. 10 indicates the co-integration of pMON128 with a disarmedTi-plasmid, thereby creating a non-tumorigenic cointegrate plasmid.

FIG. 11 is a graph comparing growth of transformed cells andnon-transformed cells on kanamycin-containing medium.

FIG. 12 represents the structure of a typical eukaryotic gene.

FIG. 13 is a flow chart representing steps of this invention, correlatedwith an example chimeric NOS-NPTII-NOS gene

FIG. 14 represents fragment HindIII-23, obtained by digesting a Tiplasmid with HindIII.

FIG. 15 represents a DNA fragment which contains a NOS promoter region,a NOS 5′ non-translated region, and the first few codons of the NOSstructural sequence.

FIG. 16 represents the cleavage of a DNA sequence at a precise location,to obtain a DNA fragment which contains a NOS promoter region andcomplete 5′ non-translated region.

FIG. 17 represents the creation of plasmids pMON1001 and pMON40, whichcontain an NPTII structural sequence.

FIG. 18 represents the insertion of a NOS promoter region into plasmidpMON40, to obtain pMON58.

FIG. 19 represents the creation of an M13 derivative designated as M-2,which contains a NOS 3′ non-translated region and poly-A signal.

FIG. 20 represents the assembly of the NOS-NPTII-NOS chimeric gene, andthe insertion of the chimeric gene into plasmid pMON38 to obtainplasmids pMON75 and pMON76.

FIG. 21 represents the creation of plasmid pMON66, which contains anNPTI gene.

FIG. 22 represents the creation of plasmid pMON73, containing a chimericNOS-NPTII sequence.

FIG. 23 represents the creation of plasmid pMON78, containing a chimericNOS-NPTI sequence.

FIG. 24 represents the creation of plasmids pMON106 and pMON107, whichcontain chimeric NOS-NPTI-NOS genes.

FIG. 25 represents the insertion of a chimeric NOS-NPTI-NOS gene intopMON120 to obtain plasmids pMON130 and pMON131.

FIG. 26 represents the structure of a DNA fragment containing a soybeanprotein (sbss) promoter.

FIG. 27 represents the creation of plasmid pMON121, containing the sbsspromoter.

FIG. 28 represents the insertion of a chimeric sbss-NPTII-NOS gene intopMON120 to create plasmids pMON141 and pMON142.

FIG. 29 represents the creation of plasmid pMON108, containing a bovinegrowth hormone structural sequence and a NOS 3′ region.

FIG. 30 represents the creation of plasmid N25-BGH, which contains theBGH-NOS sequence surrounded by selected cleavage sites.

FIG. 31 represents the insertion of a chimeric sbss-BGH-NOS gene intopMON120 to obtain plasmids pMON147 and pMON148.

FIG. 32 represents the creation of plasmid pMON149, which contains achimeric NOS-BGH-NOS gene.

FIG. 33 represents the creation of plasmid pMON8, which contains astructural sequence for EPSP synthase.

FIG. 34 represents the creation of plasmid pMON25, which contains anEPSP synthase structural sequence with several cleavage site near thestart codon.

FIG. 35 represents the creation of plasmid pMON146, which contains achimeric sequence comprising EPSP synthase and a NOS 3′ region.

FIG. 36 represents the insertion of a chimeric NOS-EPSP-NOS gene intopMON120 to obtain plasmid pMON153.

FIG. 37 represents the creation of plasmid pMON154, which contains achimeric sbss-EPSP-NOS gene.

DETAILED DESCRIPTION OF THE INVENTION

In one preferred embodiment of this invention, a variety of chimericgenes were inserted into plant cells using the steps that are summarizedon the flow chart of FIG. 1. As shown on FIG. 1, three preliminaryplasmids were prepared. Those plasmids were designated as:

1. pMON41, which contained a right border from a nopaline-type Tiplasmid, and the 5′ portion of a nopaline synthase (NOS) gene. Theconstruction of plasmid pMON41 is described below and shown in FIG. 2.

2. pMON109, which contained the 3′ portion of a NOS gene, and aselectable marker gene (spc/str) which allowed for the selection of A.tumefaciens cells having co-integrate Ti plasmids with chimeric genes.The construction of plasmid pMON109 is described below and shown inFIGS. 3, 4, and 5.

3. pMON113, which contained a region of DNA with a sequence that isidentical to the sequence within the T-DNA portion of an octopine-typeTi plasmid. This region was designated as the “left inside homology”(LIR) region. The construction of pMON113 is described below and shownin FIG. 6.

After these plasmids were assembled, each plasmid was digested byappropriate endonucleases to obtain a desired fragment. Three fragments(one from each of the three plasmids) were assembled in a tripleligation to obtain the intermediate vector, plasmid pMON120, as shown inFIG. 7.

Plasmid pMON120 plays a key role in the embodiment of this inventionthat is described in detail below. This plasmid has the followingcharacteristics:

1. pMON120 has at least three unique restriction endonuclease cleavagesites (EcoRI, ClaI, and HindIII) which allow for the convenientinsertion of any desired gene.

2. pMON120 will replicate within normal E. coli cells. However, it willnot replicate within normal Agrobacterium cells unless it co-integrateswith another plasmid, such as a Ti plasmid, which will replicate inAgrobacterium cells.

3. pMON120 carries a marker gene which codes for an enzyme which confersresistance to two antibiotics, spectinomycin (spc) and streptomycin(str). This gene, referred to as the spc/str gene, is expressed in E.coli and in A. tumefaciens, but not in plant cells. pMON120 does notcarry genes which code for resistance to ampicillin or tetracycline.

4. pMON120 carries a sequence which is homologous to a sequence withinthe T-DNA portion of an octopine-type Ti plasmid of A. tumefaciens. Thissequence is referred to as the “left inside homology” (LIH) region. Thisregion of homology promotes a crossover event whereby pMON120, or aderivative of pMON120 such as pMON128, forms a co-integrate with the Tiplasmid if the two plasmids exist inside the same A. tumefaciens cell.By definition, the “co-integrate” plasmid is formed by a singlecrossover event. It contains all DNA sequences that previously existedin either the Ti plasmid or the pMON120-derived plasmid.

5. pMON120 carries a nopaline-type T-DNA “right border,” i.e., asequence which is capable of acting as one end (designated by conventionas the right border) of a T-DNA sequence which is transferred from a Tiplasmid and inserted into the chromosome of a plant cell duringtransformation of the cell by A. tumefaciens.

6. pMON120 carries a gene (including a promoter) which codes for theexpression of an enzyme, nopaline synthase (NOS). Once introduced into aplant cell, the NOS enzyme catalyzes the production of nopaline, a typeof opine. In most types of plants, opines are non-detrimental compoundswhich accumulate at low levels; the presence of nopaline can be readilydetected in plant tissue (Otten and Schilperoort, 1978). Opine genes mayserve as useful marker genes to confirm transformation, since opines donot normally exist in untransformed plant cells. If desired, the NOSgene in pMON120 may be rendered non-functional by a variety oftechniques known to those skilled in the art. For example, a BamHIcleavage site exists within the coding portion of the NOS gene; a stopcodon or other appropriate oligonucleotide sequence could be insertedinto this site to prevent the translation of NOS.

7. The relative location of the various genes, cleavage sites, and othersequences in pMON120 is very important to the performance of thisinvention. The entire pMON120 plasmid, or its derivative plasmid such aspMON128, will be contained in the co-integrate Ti plasmid.

However, only part of the co-integrate Ti plasmid (the modified T-DNAregion) will be inserted into the plant genome. Therefore, only a partof the pMON120-derived plasmid will be inserted into the plant genome.This portion begins at the T-DNA border, and stretches in one directiononly to the region of homology. In pMON120, the NOS scorable marker, thespc/str selectable marker, and the three insertion sites are within theportion of pMON120 that would be transferred into the plant genome.However, the pBR322-derived region next to the LIH, and the PvuIcleavage site, probably would not be transferred into the plant genome.Importantly, this arrangement of pMON120 and its derivatives preventsthe transfer of more than one region of homology into the plant genome,as discussed below.

8. pMON120 is about 8 kilobases long. This is sufficiently small toallow it to accomplish all of the objectives of this invention. However,if desired, it may be made somewhat smaller by the deletion of one ormore nucleotide sequences which are not essential, using methods whichare known to those skilled in the art. Such a reduction in size mightimprove the efficiency or other characteristics of the plasmid when usedfor this invention or for other purposes, as may be determined by thoseskilled in the art.

It is recognized that a wide variety of intermediate vectors whichdiffer from pMON120 in one or more respects may be prepared and utilizedby those skilled in the art. For example, the NOS marker used forscoring transformed plant cells might be deleted, or replaced orsupplemented by a different scorable or selectable marker. One suchmarker gene might comprise an antibiotic-resistance gene such as theNOS-NPT II-NOS chimeric gene described below. As another example, thespc/str marker gene used for selecting A. tumefaciens cells withco-integrate plasmids might be deleted, or replaced or supplemented by adifferent scorable or selectable marker that is expressed inAgrobacteria. As another example, a variety of T-DNA borders (such as anopaline-type “left” border, or an octopine-type left or right border)might be utilized. Similarly, more than one border (such as two or morenopaline right borders, or one nopaline right border and one octopineright border) might be inserted into the intermediate vector, in thedesired orientation; this may increase the frequency of insertion of theT-DNA into the plant genome, as may be determined by those skilled inthe art. It is also possible to insert both left and right borders (ofany type) into an intermediate vector. It is also possible to increasethe length of the region of homology; this is likely to increase thefrequency of the desired single crossover event (Leemans et al, 1981).It is also possible to select an appropriate region of homology from anytype of desired plasmid, such as a nopaline or agropine Ti plasmid or anRi plasmid; such regions will allow the intermediate vector to form aco-integrate with any desired plasmid.

Method of Creating pMON120

Plasmid pMON120 was constructed from fragments derived from 3 otherplasmids. These three plasmids were designated as pMON41, pMON109, andpMON113. The construction of each of these three plasmids is summarizedbelow; additional information is provided in the examples.

Plasmid pMON41 contributed a nopaline-type T-DNA right border and the 5′portion of a nopaline synthase (NOS) gene to pMON120. It was created bythe following method.

A nopaline-type Ti plasmid, designated as the pTiT37 plasmid, may bedigested with the HindIII endonuclease to produce a variety offragments, including a 3.4 kb fragment which is designated as theHindIII-23 fragment. This fragment contains the entire NOS gene and theT-DNA right border. The Applicants inserted a HindIII-23 fragment into aplasmid, pBR327 (Soberon et al, 1980), which had been digested withHindIII. The resulting plasmid, designated as pMON38, was digested withboth HindIII and BamHI. This produced a 2.3 kb fragment which containsthe nopaline-type right border and the portion of a NOS gene (includingthe promoter region, the 5′ non-translated region, and part of thestructural sequence). This 2.3 kb fragment was inserted into a pBR327plasmid which had been digested with HindIII and BamHI. The resultingplasmid was designated as pMON41, as shown in FIG. 2.

A variety of strains of A. tumefaciens are publicly available from theAmerican Type Culture Collection (Rockville, Mb): accession numbers arelisted in any ATCC catalog. Each strain contains a Ti plasmid which islikely to be suitable for use in this invention, as may be determinedthrough routine experimentation by those skilled in the art.

Plasmid pMON109 contributed a spc/str selectable marker gene and the 3′portion of a NOS gene to pMON120. It was created by the followingmethod.

Plasmid pMON38 (described above and shown on FIG. 2) was digested withRsaI, which creates blunt ends as shown:5′-GTACCATG

A 1.1 kb fragment was isolated, and digested with BamHI to obtainfragments of 720 bp and 400 bp, each of which had one blunt Rsa end anda cohesive BamHI end. These fragments were added to double stranded DNAfrom a phage M13 mp 8 (Messing and Vieira, 1982) which had been digestedwith SmaI (which creates blunt ends) and BamHI. The mixture was ligated,transformed into cells and plated for recombinant phage. Recombinantphage DNA's which contained the inserted 720 bp fragment were identifiedby the size of the BamHI-SmaI insert. One of those phage DNA's wasdesignated as M-4, as shown in FIG. 3. The 720 bp insert contained the3′ non-translated region (including the poly-adenylation signalindicated in the figures by a heavy dot) of the NOS gene, as well as the3′ portion of the structural sequence of the NOS gene. The 720 bp insertis surrounded in M-4 by EcoRI and PstI cleavage sites, which werepresent in the M13 mp 8 DNA.

A bacterial transposon, designated as Tn7, is known to contain thespc/str gene, mentioned previously. The Tn7 transposon also contains agene which causes the host cell to be resistant to the antibiotictrimethoprim. The exact location and orientation of the spc/str gene andthe trimethoprim-resistance gene in Tn7, are not known. The Tn7transposon may be obtained from a variety of cell strains which arepublicly available. A strain of A. tumefaciens was isolated in which theTn7 transposon had been inserted into the Hind 111-23 region of a pTiT37plasmid. The modified pTiT37 plasmid was designated as pGV3106(Hernalsteens et al, 1980).

Plasmid pGV3106 was digested with HindIII, and the fragments wereshotgun-cloned into pBR327 plasmids which had been digested withHindIII. These plasmids were inserted into E. coli cells, and cellswhich were ampicillin-resistant (due to a pBR327 gene) andtrimethoprim-resistant (due to a Tn7 gene) were selected. The plasmidobtained from one colony was designated as pMON31. This plasmidcontained a 6 kb HindIII insert. The insert contained thespc/str-resistance gene and trimethoprim-resistance gene from Tn7, andthe 3′ portion of a NOS gene (which came from the pTiT37 plasmid).

Plasmid pMON31 was reduced in size twice. The first reduction wasperformed by digesting the plasmid with EcoRI, diluting the mixture toremove an 850 bp fragment, and religating the large fragment. Theresulting plasmid, designated as pMON53, was obtained from transformedcells selected by their resistance to ampicillin and streptomycin.Resistance to trimethoprim was not determined.

Plasmid pMON53 Was further reduced in size by digesting the plasmid withClaI, diluting the mixture to remove a 2 kb fragment, and religating thelarge fragment. The resulting 5.2 kb plasmid was designated as pMON54,as shown in FIG. 4. This plasmid contains the spc/str gene.

Plasmid pMON54 was digested with EcoRI and PstI, and a 4.8 kb fragmentcontaining the spc/str gene was isolated. M-4 DNA was digested withEcoRI and PstI, and a 740 bp fragment containing the NOS 3′non-translated region was isolated. These fragments were ligatedtogether to form pMON64. In order to be able to obtain the NOS 3′portion and the spc/str gene on a single EcoRI-BamHI fragment, theorientation of the spc/str gene was reversed by digesting pMON64 withClaI and religating the mixture. Plasmids having the desired orientationwere identified by cleavage using EcoRI and BamHI. These plasmids weredesignated as pMON109, as shown in FIG. 5.

Plasmid pMON113 contributed a region of homology to pMON120 which allowspMON120 to form a co-integrate plasmid when present in A. tumefaciensalong with a Ti plasmid. The region of homology was taken from anoctopine-type Ti plasmid. In the Ti plasmid, it is located near the leftT-DNA border, within the T-DNA portion of the Ti plasmid. This region ofhomology is designated as the “left inside homology” (L1H) region.

A region of homology may be derived from any type of plasmid capable oftransforming plant cells, such as any Ti plasmid or any Ri plasmid. Anintermediate vector can be designed which can form a co-integrateplasmid with whatever type of plasmid the region of homology was derivedfrom.

In addition, it might not be necessary for the region of homology to belocated within the T-DNA. For example, it may be possible for a regionof homology to be derived from a segment of a Ti plasmid Which containsa T-DNA border and a sequences of bases outside of the T-DNA region.Indeed, if the intermediate vector contains two appropriate T-DNAborders, it might be possible for the region of homology to be locatedentirely outside of the T-DNA region.

The Applicants obtained an E. coli culture with a pBR-derived plasmidcontaining the Bam-8 fragment of an octopine-type Ti plasmid. The Bam-8fragment, which is about 7.5 kb, contains the left border and the LIHregion of the Ti plasmid (Willmitzer et al, 1982; DeGreve et al, 1981).The Bam-8 fragment was inserted into the plasmid pBR327, which had beendigested with BamHI. The resulting plasmid was designated as pMON90, asshown in FIG. 6.

Plasmid pMON90 was digested with BglII, and a 2.6 kb fragment whichcontains the LIH region but not the left border was purified. The 2.6 kbfragment was treated with Klenow polymerase to convert the cohesive endsinto blunt ends, and the fragment was digested with HindIII to obtain a1.6 kb fragment (the desired fragment) and a 1 kb fragment. Bothfragments were mixed with a pBR322 plasmid which had been digested withPvuII and HindIII. The mixture was ligated, and inserted into E. colicells. The cells were selected for ampicillin resistance, and scored forthe presence of a SmaI site which exists on the 1.6 kb fragment but notthe 1 kb fragment. A colony having the desired plasmid was identified,and the plasmid froth this colony was designated as pMON113, asindicated by FIG. 6.

To assemble pMON120, three fragments had to be isolated. Plasmid pMON41was digested with PvuI and BamI, and a 1.5 kb fragment containing anopaline-type right border and the 5′ portion of a NOS gene wasisolated. Plasmid pMON109 was digested with BamHI and EcoRI, and a 3.4kb fragment containing a spc/str gene and the 3′ part of a NOS gene wasisolated. Plasmid pMON113 was digested with PvuI and EcoRI, and a 3.1 kbfragment containing the LIH region was isolated.

The three fragments were mixed together and ligated to form pMON120, asshown on FIG. 7. A culture of E. coli containing pMON120 has beendeposited with the American Type Culture Center. This culture has beenassigned accession number 39263.

It is recognized that a variety of different methods could be used tocreate pMON120, or any similar intermediate vector. For example, insteadof the triple ligation, it would have been possible to assemble two ofthe desired fragments in a plasmid, and insert the third fragment intothe plasmid.

Method of Using pMON120

As mentioned previously, pMON120 has three unique cleavage sites (EcoRI,ClaI, and HindIII) which are suitable for the insertion of any desiredgene. These cleavage sites are located in the portion of pMON120 thatwill be inserted into a plant genome, so the inserted gene also will beinserted into the plant genome.

A variety of chimeric genes which are capable of expressing bacterialand mammalian polypeptides in plant cells have been created by theApplicants. These chimeric genes are described in detail in a separate,simultaneously-filed application entitled “Chimeric Genes Suitable forExpression in Plant Cells,” Ser. No. 458,414. The contents of thatapplication are hereby incorporated by reference. Those chimeric genesare suitable for use in this invention. They may be inserted intopMON120 to create a derivative plasmid, which may be utilized asdescribed below.

The chimeric gene comprises a promoter region which is capable ofcausing RNA polymerase in a plant cell to create messenger RNAcorresponding to the DNA. One such promoter region comprises a nopalinesynthase (NOS) promoter region, which normally exists in certain typesof Ti plasmids in bacteria, A. tumefaciens. The NOS gene normally isinactive while contained in A. tumefaciens cells, and it becomes activeafter the Ti plasmid enters a plant cell. Other suitable promoterregions may be derived from genes which exist naturally in plant cells.

The chimeric gene also contains a sequence of bases which codes for a 5′non-translated region of mRNA which is capable of enabling or increasingthe expression in a plant cell of a structural sequence of the mRNA. Forexample, a suitable 5′ non-translated region may be taken from the NOSgene mentioned above, or from a gene which exists naturally in plantcells.

The chimeric gene also contains a desired structural sequence, i.e., asequence which is transcribed into mRNA which is capable of beingtranslated into a desired polypeptide. The structural sequence isheterologous with respect to the promoter region, and it may code forany desired polypeptide, such as a bacterial or mammalian protein. Thestructural sequence includes a start codon and a stop codon. Thestructural sequence may contain introns which are removed from the mRNAprior to translation.

If desired, the chimeric gene may also contain a DNA sequence whichcodes for a 3′ non-translated region (including a poly-adenylationsignal) of mRNA. This region may be derived from a gene which isnaturally expressed in plant cells, to help ensure proper expression ofthe structural sequence. Such genes include the NOS gene mentionedabove, as well as genes which exist naturally in plant cells.

If properly assembled and inserted into a plant genome, a chimeric geneof this invention will be expressed in the plant cell to create adesired polypeptide, such as a mammalian hormone, or a bacterial enzymewhich confers antibiotic or herbicide resistance upon the plant.

In one preferred embodiment of this invention, a chimeric gene wascreated which comprises the following DNA sequences:

1. a promoter region and a 5′ non-translated region derived from anopaline synthase (NOS) gene;

2. a structural sequence derived from a neomycin phosphotransferase II(NPT II) gene; and

3. a 3′ non-translated region derived from a NOS gene.

This chimeric NOS-NPT II-NOS gene was isolated on a DNA fragment havingEcoRI ends. This fragment was inserted into the EcoRI cleavage site ofpMON120, and the resulting plasmids (having chimeric gene inserts withopposite orientations) were designated as pMON128 and pMON129, as shownin FIG. 8. Plasmid pMON129 has two copies of the chimeric gene; this maybe a useful feature in certain types of work. Either plasmid may beutilized to transform plant cells, in the following manner. A culture ofE. coli containing pMON128 has been deposited with the American TypeCulture Collection. This culture has been assigned accession number39264.

The method used to assemble this chimeric gene is summarized in the flowchart of FIG. 13, and described in detail below and in the examples. Toassist the reader in understanding the steps of this method, variousplasmids and fragments involved in the NOS-NPTII-NOS chimeric gene arecited in parentheses in FIG. 13. However, the method of FIG. 13 isapplicable to a wide variety of other plasmids and fragments. To furtherassist the reader, the steps shown in FIG. 13 have been assigned calloutnumbers 42 et seq. These callout numbers are cited in the followingdescription. The techniques and DNA sequences of this invention arelikely to be useful in the transformation of a wide variety of plants,including any plant which may be infected by one or more strains of A.tumefaciens or A. rhizogenes.

The NOS Promoter Region and 5′ Non-Translated Region

The Applicants decided to obtain and utilize a nopaline synthase (NOS)promoter region to control the expression of the heterologous gene. TheNOS is normally carried in certain types of Ti plasmids, such as pTiT37.Sciaky et al, 1978. The NOS promoter is normally inactive while in an A.tumefaciens cell. The entire NOS gene, including the promoter and theprotein coding sequence, is within the T-DNA portion of a Ti plasmidthat is inserted into the chromosomes of plant cells when a plantbecomes infected and forms a crown gall tumor. Once inside the plantcell, the NOS promoter region directs RNA polymerase within a plant cellto transcribe the NOS protein coding sequence into mRNA, which issubsequently translated into the NOS enzyme.

The boundaries between the different parts of a promoter region (shownin FIG. 12 as association region 2, intervening region 4, transcriptioninitiation sequence 6, and intervening region 8), and the boundarybetween the promoter region and the 5′ non-translated region, are notfully understood. The Applicants decided to utilize the entire promoterregion and 5′ non-translated region from the NOS gene, which is known tobe expressed in plant cells. However, it is entirely possible that oneor more of these sequences might be modified in various ways, such asalteration in length or replacement by other sequences. Suchmodifications in promoter regions and 5′ non-translated regions havebeen studied in bacterial cells (see, e.g., Roberts et al 1979) andmammalian cells (see, e.g., McKnight, 1982). By utilizing themethodology taught by this invention, it is now possible to study theeffects of modifications to promoter regions and 5′ non-translatedregions on the expression of genes in plant cells. It may be possible toincrease the expression of a gene in a plant cell by means of suchmodifications. Such modifications, if performed upon chimeric genes ofthis invention, are within the scope of this invention.

A nopaline-type tumor-inducing plasmid, designated as pTiT37, wasisolated from a strain of A. tumefaciens using standard procedures(Currier and Nester, 1976). It was digested with the endonucleaseHindIII which produced numerous fragments. These fragments wereseparated by size on a gel, and one of the fragments was isolated andremoved from the gel. This fragment was designated as the HindIII-23fragment, because it was approximately the 23rd largest fragment fromthe Ti plasmid; it is approximately 3400 base pairs (bp) in size, alsoreferred to as 3.4 kilobases (kb). From work by others (see, e.g.,Hernalsteens et al, 1980), it was known that the HindIII-23 fragmentcontained the entire NOS gene, including the promoter region, a 5′non-translated region, a structural sequence with a start codon and astop codon, and a 3′ non-translated region. The HindIII-23 fragment isshown in FIG. 14.

By means of various cleavage and sequencing experiments, it wasdetermined that the HindIII-23 fragment could be digested by anotherendonuclease, Sau3a, to yield a fragment, about 350 bp in size, whichcontains the entire NOS promoter region, the 5′ non-translated region,and the first few codons of the NOS structural sequence. This fragmentwas sequenced, and the base sequence is represented in FIG. 15. Thestart codon (ATG) of the NOS structural sequence begins at base pair 301within the 350 bp fragment. The Applicants decided to cleave thefragment between base pairs 300 and 301; this would provide them with afragment about 300 base pairs long containing a NOS promoter region andthe entire 5′ non-translated region but with no translated bases. Tocleave the 350 bp fragment at precisely the right location, theApplicants obtained an M13 clone designated as S1A, and utilized theprocedure described below.

To create the S1A clone, Dr. Michael Bevan of Washington Universityconverted the 350 bp Sau3a fragment into a single strand of DNA. Thiswas done by utilizing a virus vector, designated as the M13 mp 2 phage,which goes through both double-stranded (ds) and single-stranded (ss)stages in its life cycle (Messing et al, 1981). The ds 350 bp fragmentwas inserted into the double-stranded replicative form DNA of the M13 mp2, which had been cleaved with BamHI. The two fragments were ligated,and used to infect E. coli cells. The ds DNA containing the 350 bpinserted fragment subsequently replicated, and one strand (the viralstrand) was encapsulated by the M13 viral capsid proteins. In one clone,designated the S1A, the orientation of the 350 bp fragment was such thatthe anti-sense strand (containing the same sequence as the mRNA) of theNOS gene was carried in the viral strand. Viral particles released frominfected cells were isolated, and provided to the Applicants.

Single stranded S1A DNA, containing the anti-sense 350 bp fragment withthe NOS promoter region, was isolated from the viral particles andsequenced. A 14-mer oligonucleotide primer was synthesized, usingpublished procedures (Beaucage and Carruthers, 1981, as modified byAdams et al, 1982). This 14-mer was designed to be complementary tobases 287 through 300 of the 350 bp fragment, as shown on FIG. 15.

The 5′ end of the synthetic primer was radioactively labelled with ³²P;this is represented in the figures by an asterisk

Copies of the primer were mixed with copies of the single-stranded S1ADNA containing the anti-sense strand of the 350 bp fragment. The primerannealed to the desired region of the S1A DNA, as shown at the top ofFIG. 5. After this occurred, Klenow DNA polymerase and a controlledquantity of unlabelled deoxy-nucleoside triphosphates (dNTP's), A, T, C,and G, were added. Klenow polymerase added nucleotides to the 3′(unlabelled) end of the primer, but not to the 5′ (labelled) end. Theresult, as shown in FIG. 16, was a circular loop of single-stranded DNA,part of which was matched by a second strand of DNA. The 5′ end of thesecond strand was located opposite base #300 of the Sau3a insert

The partially double-stranded DNA was then digested by a thirdendonuclease, HaeIII, which can cleave both single-stranded anddouble-stranded DNA. HaeIII cleavage sites were known to exist inseveral locations outside the 350 bp insert, but none existed inside the350 bp insert. This created a fragment having one blunt end, and one 3′overhang which started at base #301 of the Sau3a insert.

The HaeIII fragment mixture was treated with T4 DNA polymerase andunlabelled dNTP's. This caused the single stranded portion of the DNA,which extended from base #301 of the Sau3a insert to the closest HaeIIIcleavage site, to be removed from the fragment. In this manner, the ATGstart codon was removed from base pair #300, leaving a blunt enddouble-stranded fragment which was approximately 550 bp long.

The mixture was then digested by a fourth endonuclease EcoRI, whichcleaved the 550 bp fragment at a single site outside the NOS promoterregion. The fragments were then separated by size on a gel, and theradioactively-labelled fragment was isolated. This fragment containedthe entire NOS promoter region and 5′ non-translated region. It had oneblunt end with a sequence of5′ . . . CTGCA. . . GACGTand one cohesive end (at the EcoRI site) with a sequence of The shorterstrand was about 308 bp long.

The foregoing steps are represented in FIG. 2 as steps 42, 44, and 46.

This fragment was inserted into pMON40 (which is described below) toobtain pMON58, as shown on FIG. 13.

Creation of Plasmid with NPTII Gene (pMON40)

A bacterial transposon, designated as Tn5, is known to contain acomplete NPTII gene, including promoter region, structural sequence, and3′ non-translated region. The NPTII enzyme inactivates certainaminoglycoside antibiotics, such as kanamycin, neomycin, and G418; seeJimenez and Davies, 1980. This gene is contained within a 1.8 kbfragment, which can be obtained by digesting phage lambda bbkan-1 DNA(D. Berg et al, 1975) with two endonucleases, HindIII and BamHI. Thisfragment was inserted into a common laboratory plasmid, pBR327, whichhad been digested by HindIII and BamHI. As shown in FIG. 17, theresulting plasmid was designated as pMON1001, which was about 4.7 kb.

To reduce the size of the DNA fragment which carried the NPTIIstructural sequence, the Applicants eliminated about 500 bp from thepMON1001 plasmid, in the following manner. First, they digested pMON1001at a unique SmaI restriction site which was outside of the NPTII gene.Next, they inserted a 10-mer synthetic oligonucleotide linker,5′CCGGATCCGG,GGCCTAGGCCinto the SmaI cleavage site. This eliminated the SmaI cleavage site andreplaced it with a BamHI cleavage site. A second BamHI cleavage sitealready existed, about 500 bp from the new BamHI site. The Applicantsdigested the plasmid with BamHI, separated the 500 bp fragment from the4.2 kb fragment, and circularized the 4.2 kb fragment. The resultingplasmids were inserted into E. coli, which were then selected forresistance to ampicillin and kanamycin. A clonal colony of E. coli wasselected; these cells contained a plasmid which was designated aspMON40, as shown in FIG. 17.

The foregoing steps are represented in FIG. 13 as steps 48 and 50.

Insertion of NOS Promoter into Plasmid pMON40

-   -   The Applicants deleted the NPTII promoter from pMON40, and        replaced it with the NOS promoter fragment described previously,        by the following method, shown on FIG. 18.

Previous cleavage and sequencing experiments (Rao and Rogers, 1979;Auerswald et al, 1980) indicated that a BglII cleavage site existed inthe NPTII gene between the promoter region and the structural sequence.Plasmid pMON40 was digested with BglII. The cohesive ends were thenfilled in by mixing the cleaved plasmid with Klenow polymerase and thefour dNTP's, to obtain the following blunt ends:

5′ - AGATC GATCT- - TCTAG CTAGA-5′

The polymerase and dNTP's were removed, and the cleaved plasmid was thendigested with EcoRI. The smaller fragment which contained the NPTIIpromoter region was removed, leaving a large fragment with one EcoRI endand one blunt end. This large fragment was mixed with the 308 bpfragment which contained the NOS promoter, described previously andshown on FIG. 5. The fragments were ligated, and inserted into E. coli.E. coli clones were selected for ampicillin resistance. Replacement ofthe NPTII promoter region (a bacterial promoter) with the NOS promoterregion (which is believed to be active only in plant cells) caused theNPTII structural sequence to become inactive in E. coli. Plasmids from36 kanamycin-sensitive clones were obtained; the plasmid from one clone,designated as pMON58, was utilized in subsequent work.

The foregoing steps are represented in FIG. 13 as steps 52 and 54.

Plasmid pMON58 may be digested to obtain a 1.3 kb EcoRI-BamHI fragmentwhich contains the NOS promoter region, the NOS 5′ non-translatedregion, and the NPTII structural sequence. This step is represented inFIG. 13 as step 56.

Insertion of NOS 3′ Sequence into NPTII Gene

As mentioned above in “Background Art”, the functions of 3′non-translated regions in eucaryotic genes are not fully understood.However, they are believed to contain at least one important sequence, apoly-adenylation signal.

It was suspected by the Applicants that a gene having a bacterial 3′non-translated region might not be expressed as effectively in a plantcell as the same gene having a 3′ non-translated region from a gene,such as NOS, which is known to be expressed in plants.

Therefore, the Applicants decided to add a NOS 3′ non-translated regionto the chimeric gene, in addition to the NPTII 3′ non-translated regionalready present. Whether a different type of 3′ non-translated region(such as a 3′ region from an octopine-type or agropine-type Ti plasmid,or a 3′ region from a gene that normally exists in a plant cell) wouldbe suitable or preferable for use in any particular type of chimericgene, for use in any specific type of plant cell, may be determined bythose skilled in the art through routine experimentation using themethod of this invention. Alternately, it is possible, using the methodsdescribed herein, to delete the NPTII or other existing 3′non-translated region and replace it with a desired 3′ non-translatedregion that is known to be expressed in plant cells.

Those skilled in the art may also determine through routineexperimentation whether the 3′ non-translated region that naturallyfollows a structural sequence that is to be inserted into a plant cellwill enhance the efficient expression of that structural sequence inthat type of plant cell. If so, then the steps required to insert adifferent 3′ non-translated region into the chimeric gene might not berequired in order to perform the method of this invention.

In order to obtain a DNA fragment containing a NOS 3′ non-translatedregion appropriate for joining to the NPTII structural sequence frompMON58 (described previously), the Applicants utilized a 3.4 kbHindIII-23 fragment from a Ti plasmid, shown on FIG. 14. This 3.4 kbfragment was isolated and digested with BamHI to obtain a 1.1 kbBamHI-HindIII fragment containing a 3′ portion of the NOS structuralsequence (including the stop codon), and the 3′ non-translated region ofthe NOS gene (including the poly-adenylation signal). This 1.1 kbfragment was inserted into a pBR327 plasmid which had been digested withHindIII and BamHI. The resulting plasmid was designated as pMON42, asshown on FIG. 19.

Plasmid pMON42 was digested with BamHI and RsaI, and a 720 bp fragmentcontaining the desired NOS 3′ non-translated region was purified on agel. The 720 bp fragment was digested with another endonuclease, MboI,and treated with the large fragment of E. coli DNA polymerase I. Thisresulted in a 260 bp fragment with MboI blunt ends, containing a largepart of the NOS 3′ non-translated region including the poly-A signal.

The foregoing procedure is represented in FIG. 13 by step 58. However,it is recognized that alternate means could have been utilized; forexample, it might have been possible to digest the HindIII-23 fragmentdirectly with MboI to obtain the desired 260 bp fragment with the NOS 3′non-translated region.

Assembly of Chimeric Gene

To complete the assembly of the chimeric gene, it was necessary toligate the 260 bp MboI fragment (which contained the NOS 3′non-translated region) to the 1.3 kb EcoRI-BamHI fragment from pMON58(which contained the NOS promoter region and 5′ non-translated regionand the NPTII structural sequence). In order to facilitate this ligationand control the orientation of the fragments, the Applicants decided toconvert the MboI ends of the 260 bp fragment into a BamHI end (at the 5′end of the fragment) and an EcoRI end (at the 3′ end of the fragment).In order to perform this step, the Applicants used the following method.

The 260 bp MboI fragment, the termini of which had been converted toblunt ends by Klenow polymerase, was inserted into M13 mp 8 DNA at aSmaI cleavage site. The SmaI site is surrounded by a variety of othercleavage sites present in the M13 mp 8 DNA, as shown in FIG. 19. TheMboI fragment could be inserted into the blunt SmaI ends in eitherorientation. The orientation of the MboI fragments in different cloneswere tested, using HinfI cleavage sites located assymetrically withinthe MboI fragment. A clone was selected in which the 3′ end of the NOS3′ non-translated region was located near the EcoRI cleavage site in theM13 mp 8 DNA. This clone was designated as the M-2 clone, as shown inFIG. 19.

Replicative form (double stranded) DNA from the M-2 clone was digestedby EcoRI and BamHI and a 280 bp fragment was isolated. Separately,plasmid pMON58 was digested by EcoRI and BamHI, and a 1300 bp fragmentwas isolated. The two fragments were ligated, as shown in FIG. 20, tocomplete the assembly of a NOS-NPTII-NOS chimeric gene having EcoRIends.

There are a variety of ways to control the ligation of the twofragments. For example, the two EcoRI-BamHI fragments could be joinedtogether with DNA ligase and cleaved with EcoRI. After inactivation ofEcoRI, a vector molecule having EcoRI ends that were treated with calfalkaline phosphatase (CAP) may be added to the mixture. The fragments inthe mixture may be ligated in a variety of orientations. The plasmidmixture is used to transform E. coli, and cells having plasmids with thedesired orientation are selected or screened, as described below.

A plasmid, designated as pMON38, was created by insertion of theHindIII-23 fragment (from Ti plasmid pTiT37) into the HindIII cleavagesite of the plasmid pBR327. Plasmid pMON38 contains a unique EcoRI site,and an ampicillin-resistance gene which is expressed in E. coli. PlasmidpMON38 was cleaved with EcoRI and treated with alkaline phosphatase toprevent it from re-ligating to itself. U.S. Pat. No. 4,264,731 (Shine,1981). The resulting fragment was mixed with the 1300 bp NOS-NPTIIfragment from pMON58, and the 280 bp NOS fragment from M-2, which hadbeen ligated and EcoRI-cleaved as described in the previous paragraph.The fragments were ligated, and inserted into E. coli. The E. coli cellswhich had acquired intact plasmids with ampicillin-resistance genes wereselected on plates containing ampicillin. Several clones were selected,and the orientation of the inserted chimeric genes was evaluated bymeans of cleavage experiments. Two clones having plasmids carryingNOS-NPTII-NOS inserts with opposite orientations were selected anddesignated as pMON75 and pMON76, as shown in FIG. 20. The chimeric genemay be isolated by digesting either pMON75 or pMON76 with EcoRI andpurifying a 1580 bp fragment.

The foregoing procedure is represented on FIG. 13 by step 60.

This completes the discussion of the NOS-NPTII-NOS chimeric gene.Additional information on the creation of this gene is provided in theExamples. A copy of this chimeric gene is contained in plasmid pMON128;it may be removed from pMON128 by digestion with EcoRI. A culture of E.coli containing pMON128 has been deposited with the American TypeCulture Collection; this culture has been assigned accession number39264.

To prove the utility of this chimeric gene, the Applicants inserted itinto plant cells. The NPTII structural sequence was expressed in theplant cells, causing them and their descendants to acquire resistance toconcentrations of kanamycin which are normally toxic to plant cells.

In an alternate preferred embodiment of this invention, a chimeric genewas created comprising (1) a NOS promoter region and 5′ non-translatedregion, (2) a structural sequence which codes for NPTI, and (3) a NOS 3′non-translated region. NPTI and NPTII are different and distinct enzymeswith major differences in their amino acid sequences and substratespecificities. See, e.g., E. Beck et al, 1982. The relative stabilitiesand activities of these two enzymes in various types of plant cells arenot yet fully understood, and NPTI may be preferable to NPTII for use incertain types of experiments and plant transformations.

A 1200 bp fragment containing an entire NPTI gene was obtained bydigesting pACYC177 (Chang and Cohen, 1978) with the endonuclease, AvaII.The Avail termini were converted to blunt ends with Klenow polymerase,and converted to BamHI termini using a synthetic linker. This fragmentwas inserted into a unique BamHI site in a pBR327-derived plasmid, asshown in FIG. 21. The resulting plasmid was designated as pMON66.

Plasmid pMON57 (a deletion derivative of pBR327, as shown in FIG. 21)was digested with AvaII. The 225 bp fragment of pMON57 was replaced bythe analogous 225 bp AvaII fragment taken from plasmid pUC8 (Vieira andMessing, 1982), to obtain a derivative of pMON57 with no PstI cleavagesites. This plasmid was designated as pMON67.

Plasmid pMON58 (described previously and shown in FIG. 18) was digestedwith EcoRI and BamHI to obtain a 1300 bp fragment carrying the NOSpromoter and the NPTII structural sequence.

This fragment was inserted into pMON67 which had been digested withEcoRI and BamHI. The resulting plasmid was designated as pMON73, asshown in FIG. 22.

pMON73 was digested with PstI and BamHI, and a 2.4 kb fragment wasisolated containing a NOS promoter region and 5′ non-translated region.Plasmid pMON66 (shown on FIG. 21) was digested with XhoI and BamHI toyield a 950 bp fragment containing the structural sequence of NPTI. Thisfragment lacked about 30 nucleotides at the 5′ end of the structuralsequence. A synthetic linker containing the missing bases, havingappropriate PstI and XhoI ends, was created. The pMON73 fragment, thepMON66 fragment, and the synthetic linker were ligated together toobtain plasmid pMON78, as shown in FIG. 13. This plasmid contains theNOS promoter region and 5′ non-translated region adjoined to the NPTIstructural sequence. The ATG start codon was in the same position thatthe ATG start codon of the NOS structural sequence had occupied.

Plasmid pMON78 was digested with EcoRI and BamHI to yield a 1300 bpfragment carrying the chimeric NOS-NPTI regions. Double-stranded DNAfrom the M-2 clone (described previously and shown on FIG. 20) wasdigested with EcoRI and BamHI, to yield a 280 bp fragment carrying a NOS3′ non-translated region with a poly-adenylation signal. The twofragments described above were ligated together to create theNOS-NPTI-NOS chimeric gene, which was inserted into plasmid pMON38(described above) which had been digested with EcoRI. The two resultingplasmids, having chimeric gene inserts with opposite orientations, weredesignated as pMON106 and pMON107, as shown in FIG. 24.

Either of plasmids pMON106 or pMON107 may be digested with EcoRI toyield a 1.6 kb fragment containing the chimeric NOS-NPTI-NOS gene. Thisfragment was inserted into plasmid pMON120 which had been digested withEcoRI and treated with alkaline phosphatase. The resulting plasmids,having inserts with opposite orientations, were designated as pMON130and pMON131, as shown on FIG. 25.

The NOS-NPTI-NOS chimeric gene was inserted into plant cells, whichacquired resistance to kanamycin. This demonstrates expression of thechimeric gene in plant cells.

Creation of Chimeric Gene with Soybean Promoter

In an alternate preferred embodiment of this invention, a chimeric genewas created comprising (1) a promoter region and 5′ non-translatedregion taken from a gene which naturally exists in soybean; this genecodes for the small subunit of ribulose-1,5-bis-phosphate carboxylase(sbss for soybean small subunit); (2) a structural sequence which codesfor NPTII, and (3) a NOS 3′ non-translated region.

The sbss gene codes for a protein in soybean leaves which is involved inphotosynthetic carbon fixation. The sbss protein is the most abundantprotein in soybean leaves (accounting for about 10% of the total leafprotein), so it is likely that the sbss promoter region causes prolifictranscription.

There are believed to be approximately six genes encoding the ssRuBPCase protein in the soybean genome. One of the members of the ssRuBPCase gene family, SRS1, which is highly transcribed in soybeanleaves, has been cloned and characterized. The promoter region, 5′nontranslated region, and a portion of the structural sequence arecontained on a 2.1 kb EcoRI fragment that was subcloned into the EcoRIsite of plasmid pBR325 (Bolivar, 1978). The resultant plasmid, pSRS2.1,was a gift to Monsanto Company from Dr. R. B. Meagher, University ofGeorgia, Athens, Ga. The 2.1 kb EcoRI fragment from pSRS2.1 is shown onFIG. 26.

Plasmid pSRS2.1 was prepared from dam—E. coli cells, and cleaved withMboI to obtain an 800 bp fragment. This fragment was inserted intoplasmid pKC7 (Rao and Rogers, 1979) which had been cleaved with BglII.The resulting plasmid was designated as pMON121, as shown on FIG. 27.

Plasmid pMON121 was digested with EcoRI and Bcl1, and a 1200 bp fragmentcontaining the sbss promoter region was isolated. Separately, plasmidpMON75 (described previously and shown on FIG. 20) was digested withEcoRI and BglII, and a 1250 bp fragment was isolated, containing a NPTIIstructural sequence and a NOS 3′ non-translated region. The twofragments were ligated at the compatible Bcl1/BglII overhangs, to createa 2450 bp fragment containing sbss-NPTII-NOS chimeric gene. Thisfragment was inserted into pMON120 which had been cleaved with EcoRI, tocreate two plasmids having chimeric gene inserts with oppositeorientations, as shown in FIG. 28. The plasmids were designated aspMON141 and pMON142.

The sbss-NPTII-NOS chimeric genes were inserted into several types ofplant cells, causing the plant cells to acquire resistance to kanamycin.

This successful transformation proved that a promoter region from onetype of plant can cause the expression of a gene within plant cells froman entirely different genus, family, and order of plants.

The chimeric sbss-NPTII-NOS gene also had another significant feature.Sequencing experiments indicated that the 800 bp MboI fragment containedthe ATG start codon of the sbss structural sequence. Rather than removethis start codon, the Applicants decided to insert a stop codon behindit in the same reading frame.

This created a dicistronic mRNA sequence, which coded for a truncatedamino portion of the sbss polypeptide and a complete NPTII polypeptide.Expression of the NPTII polypeptide was the first proof that adicistronic mRNA can be translated within plant cells.

The sbss promoter is contained in plasmid pMON154, described below. Aculture of E. coli containing this plasmid has been deposited with theAmerican Type Culture Center. This culture has been assigned accessionnumber 39265.

Creation of BGH Chimeric Genes

-   -   In an alternate preferred embodiment of this invention, a        chimeric gene was created comprising (1) a sbss promoter region        and 5′ non-translated region, (2) a structural sequence which        codes for bovine growth hormone (BGH) and (3) a NOS 3′        non-translated region. This chimeric gene was created as        follows.

A structural sequence which codes for the polypeptide, bovine growthhormone, (see, e.g., Woychik et al, 1982) was inserted into apBR322-derived plasmid. The resulting plasmid was designated as plasmidCP-1. This plasmid was digested with EcoRI and HindIII to yield a 570 bpfragment containing the structural sequence. Double stranded M-2 RF DNA(described previously and shown in FIG. 19) was cleaved with EcoRI andHindIII to yield a 290 bp fragment which contained the NOS 3′non-translated region with a poly-adenylation signal. The two fragmentswere ligated together and digested with EcoRI to create an 860 base pairfragment with EcoRI ends, which contained a BGH-coding structuralsequence joined to the NOS 3′ non-translated region. This fragment wasintroduced into plasmid pMON38, which had been digested with EcoRI andtreated with alkaline phosphatase, to create a new plasmid, designatedas pMON108, as shown in FIG. 29.

A unique BglII restriction site was introduced at the 5′ end of the BGHstructural sequence by digesting pMON 108 with EcoRI to obtain the 860bp fragment, and using Klenow polymerase to create blunt ends on theresulting EcoRI fragment. This fragment was ligated into plasmid N25 (aderivative of pBR327 containing a synthetic linker carrying BglII andXbaI cleavage sites inserted at the BamHI site), which had been cleavedwith XbaI and treated with Klenow polymerase to obtain blunt ends (N25contains a unique BglII site located 12 bases from the XbaI site). Theresulting plasmid, which contained the 860 bp BGH-NOS fragment in theorientation shown in FIG. 30, was designated as plasmid N25-BGH. Thisplasmid contains a unique BglII cleavage site located about 25 basesfrom the 5′ end of the BGH structural sequence.

Plasmid N25-BGH prepared from dam—E. coli cells was digested with BglIIand ClaI to yield an 860 bp fragment which contained the BGH structuralsequence joined to the NOS 3′ non-translated region. Separately, plasmidpMON121 (described previously and shown in FIG. 27) was prepared fromdam—E. coli cells and was digested with ClaI and BclI to create an 1100bp fragment which contained the sbss promoter region. The fragments wereligated at their compatible BclI/BglII overhangs, and digested with ClaIto yield a ClaI fragment of about 2 kb containing the chimericsbss-BGH-NOS gene. This fragment was inserted into pMON 120 (describedpreviously and shown in FIG. 8) which had been digested with ClaI. Theresulting plasmids, containing the inserted chimeric gene in oppositeorientations were designated pMON 147 and pMON 148, as shown in FIG. 31.

An alternate chimeric BGH gene was created which contained (1) a NOSpromoter region and 5′ non-translated region, (2) a structural sequencewhich codes for BGH, and (3) a NOS 3′ non-translated region, by thefollowing method, shown in FIG. 32.

Plasmid pMON76 (described above and shown in FIG. 20) was digested withEcoRI and BglII to obtain a 308 bp fragment containing a NOS promoterregion and 5′ non-translated region. Plasmid N25-BGH prepared fromdam—E. coli cells (described above and shown in FIG. 30) was digestedwith BglII and ClaI to obtain a 900 bp fragment containing a BGHstructural sequence and a NOS 3′ non-translated region. These twofragments were ligated together to obtain a chimeric NOS-BGH-NOS gene ina fragment with EcoRI and ClaI ends. This fragment was ligated with an 8kb fragment obtained by digesting pMON120 with EcoRI and ClaI. Theresulting plasmid, designated as pMON149, is shown in FIG. 32.

Creation of Chimeric NOS-EPSP-NOS Gene

In an alternate preferred embodiment, a chimeric gene was createdcomprising (1) a NOS promoter region and 5′ non-translated region, (2) astructural sequence which codes for the E. coli enzyme,5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) and(3) a NOS 3′ non-translated region.

EPSP synthase is believed to be the target enzyme for the herbicide,glyphosate, which is marketed by Monsanto Company under the registeredtrademark, “Roundup.” Glyphosate is known to inhibit EPSP synthaseactivity (Amrhein et al, 1980), and amplification of the EPSP synthasegene in bacteria is known to increase their resistance to glyphosate.Therefore, increasing the level of EPSP synthase activity in plants mayconfer resistance to glyphosate in transformed plants. Since glyphosateis toxic to most plants, this provides for a useful method of weedcontrol. Seeds of a desired crop plant which has been transformed toincrease EPSP synthase activity may be planted in a field. Glyphosatemay be applied to the field at concentrations which will kill allnon-transformed plants, leaving the non-transformed plants unharmed.

An EPSP synthase gene may be isolated by a variety of means, includingthe following. A lambda phage library may be created which carries avariety of DNA fragments produced by HindIII cleavage of E. coli DNA.See, e.g., Maniatis et al, 1982.

The EPSP synthase gene is one of the genes which are involved in theproduction of aromatic amino acids. These genes are designated as the“aro” genes; EPSP synthase is designated as aroA. Cells which do notcontain functional aro genes are designated as aro-cells. Aro− cellsmust normally be grown on media supplemented by aromatic amino acids.See Pittard and Wallis, 1966.

Different lambda phages which carry various HindIII fragments may beused to infect mutant E. coli cells which do not have EPSP synthasegenes. The infected aro− cells may be cultured on media which does notcontain the aromatic amino acids, and transformed aro+ clones which arecapable of growing on such media may be selected. Such clones are likelyto contain the EPSP synthase gene. Phage particles may be isolated fromsuch clones, and DNA may be isolated from these phages. The phage DNAmay be cleaved with one or more restriction endonucleases, and by agradual process of analysis, a fragment which contains the EPSP synthasegene may be isolated.

Using a procedure similar to the method summarized above, the Applicantsisolated an 11 kb HindIII fragment which contained the entire E. coliEPSP synthase gene. This fragment was digested with BglII to produce a3.5 kb HindIII-BglII fragment which contained the entire EPSP synthasegene. This 3.5 kb fragment was inserted into plasmid pKC7 (Rao andRogers, 1979) to produce plasmid pMON4, which is shown in FIG. 33.

Plasmid pMON4 was digested with ClaI to yield a 2.5 kb fragment whichcontained the EPSP synthase structural sequence. This fragment wasinserted into pBR327 that had been digested with ClaI, to create pMON8,as shown in FIG. 33.

pMON8 was digested with BamHI and NdeI to obtain a 4.9 kb fragment. Thisfragment lacked about 200 nucleotides encoding the amino terminus of theEPSP synthase structural sequence.

The missing nucleotides were replaced by ligating a HinfI/NdeI fragment,obtained from pMON8 as shown in FIG. 34, together with a syntheticoligonucleotide sequence containing (1) the EPSP synthase start codonand the first three nucleotides, (2) a unique BglII site, and (3) theappropriate BamHI and HinfI ends. The resulting plasmid, pMON25,contains an intact EPSP synthase structural sequence with unique BamHIand BglII sites positioned near the start codon.

Double stranded M-2 DNA (described previously and shown in FIG. 19) wasdigested with HindIII and EcoRI to yield a 290 bp fragment whichcontains the NOS 3′ non-translated region and poly-adenylation signal.This fragment was introduced into a pMON25 plasmid that had beendigested with EcoRI and HindIII to create a plasmid, designated aspMON146 (shown in FIG. 35) which contains the EPSP structural sequencejoined to the NOS 3′ non-translated region.

pMON146 was cleaved with ClaI and BglII to yield a 2.3 kb fragmentcarrying the EPSP structural sequence joined to the NOS 3′non-translated region. pMON76 (described previously and shown in FIG.20) was digested with BglII and EcoRI to create a 310 bp fragmentcontaining the NOS promoter region and 5′ non-translated region. Theabove fragments were mixed with pMON 120 (described previously and shownin FIG. 8) that had been digested with ClaI and EcoRI, and the mixturewas ligated. The resulting plasmid, designated pMON153, is shown in FIG.36. This plasmid contains the chimeric NOS-EPSP-NOS gene.

A plasmid containing a chimeric sbss-EPSP-NOS gene wa prepared in thefollowing manner, shown in FIG. 37. Plasmid pMON146 (describedpreviously and shown in FIG. 35) was digested with ClaI and BglII, and a2.3 kb fragment was purified. This fragment contained the EPSP synthasestructural sequence coupled to a NOS 3′ non-translated region with apoly-adenylation signal. Plasmid pMON121 (described above and shown inFIG. 27) was digested with ClaI and BclI and a 1.1 kb fragment waspurified. This fragment contains an sbss promoter region and 5′non-translated region. The two fragments were mixed and ligated with T4DNA ligase and subsequently digested with ClaI. This created a chimericsbss-EPSP-NOS gene, joined through compatible BglII and BclI termini.This chimeric gene with ClaI termini was inserted into plasmid pMON120which had been digested with ClaI and treated with calf alkalinephosphatase (CAP). The mixture was ligated with T4 DNA ligase. Theresulting mixture of fragments and plasmids was used to transform E.coli cells, which were selected for resistance to spectinomycin. Acolony of resistant cells was isolated, and the plasmid in this colonywas designated as pMON154, as shown in FIG. 37.

A culture of E. coli containing pMON154 has been deposited with theAmerican Type Culture Center. This culture has been assigned accessionnumber 39265.

Plasmid pMON128 (or any other plasmid derived by inserting a desiredgene into pMON120) is inserted into a microorganism which contains anoctopine-type Ti plasmid (or other suitable plasmid). Suitablemicroorganisms include A. tumefaciens and A. rhizogenes which carry Tior Ri plasmids. Other microorganisms which might also be useful for usein this invention include other species of Agrobacterium, as well asbacteria in the genus Rhizobia. The suitability of these cells, or ofany other cells known at present or hereafter discovered or created, foruse in this invention may be determined through routine experimentationby those skilled in the art.

The plasmid may be inserted into the microorganism by any desiredmethod, such as transformation (i.e., contacting plasmids with cellsthat have been treated to increase their uptake of DNA) or conjugationwith cells that contain the pMON128 or other plasmids.

The inserted plasmid (such as pMON128) has a region which is homologousto a sequence within the Ti plasmid. This “LIH” region of homologyallows a single crossover event whereby pMON128 and an octopine-type Tiplasmid combine with each other to form a co-integrate plasmid. See,e.g., Stryer, supra, at p. 752-754. Normally, this will occur within theA. tumefaciens cell after pMON128 has been inserted into the cell.Alternately, the co-integrate plasmid may be created in a different typeof cell or in vitro, and then inserted into an A. tumefaciens or othertype of cell which can transfer the co-integrate plasmid into plantcells.

The inserted plasmid, such as pMON128, combines with the Ti plasmid inthe manner represented by FIG. 9 or 10, depending upon what type of Tiplasmid is involved.

In FIG. 9, item 2 represents the T-DNA portion of an octopine-type Tiplasmid. Item 4 represents the inserted plasmid, such as pMON128. Whenthese two plasmids co-exist in the same cell, a crossover event canoccur which results in the creation of co-integrate plasmid 6.

Co-integrate plasmid 6 has one left border 8, and two right borders 10and 12. The two right borders are designated herein as the “proximal”right border 10 (the right border closest to left border 8), and the“distal” right border 12 (the right border that is more distant fromleft border 8. Proximate right border 10 was carried by plasmid 4; thedistal right border was contained on Ti plasmid 2 before co-integration.

A culture of A. tumefaciens GV3111 containing a co-integrate plasmidformed by pMON1-28 and wild-type Ti plasmid pTiB653 has been depositedwith the American Type Culture Center. This culture has been assignedaccession number 39266.

When co-integrate Ti plasmid 6, shown in FIG. 9, is inserted into aplant cell, either of two regions of DNA may enter the plant genome,T-DNA region 14 or T-DNA region 16.

T-DNA region 14 is bounded by left border 8 and proximate right border10. Region 14 contains the chimeric gene and any other genes containedin plasmid 4, such as the spc/str selectable marker and the NOS scorablemarker. However, region 14 does not contain any of the T-DNA genes whichwould cause crown gall disease or otherwise disrupt the metabolism orregenerative capacity of the plant cell.

T-DNA region 16 contains left border 8 and both right borders 10 and 12.This segment of T-DNA contains the chimeric gene and any other genescontained in plasmid 4. However, T-DNA region 16 also contains the T-DNAgenes which are believed to cause crown gall disease.

Either of the foregoing T-DNA segments, Region 14 or Region 16, might betransferred to the plant DNA. This is presumed to occur at a 50-50probability for any given T-DNA transfer. This is likely to lead to amixture of transformed cells, some of which are tumorous and some orwhich are non-tumorous. It is possible to isolate and cultivatenon-tumorous cells from the mixture, as described in the examples.

An alternate approach has also been developed which avoids the need forisolating tumorous from non-tumorous cells. Several mutant strains of A.tumefaciens have been isolated which are incapable of causing crown galldisease. Such strains are usually referred to as “disarmed” Ti plasmids.A Ti or Ri plasmid may be disarmed by one or more of the following typesof mutations:

1. Removal or inactivation of one of the border regions. One suchdisarmed octopine plasmid, which has a left border but not a rightborder, is designated as pAL4421; this plasmid is contained in A.tumefaciens strain LBA4421 (Ooms et al, 1982; Garfinkel et al, 1981).

2. Removal or inactivation of the one or more of the “tumor morphology”genes, designated as the tmr and tms genes. See, e.g., Leemans et al,1982.

Various other types of disarmed plant tumor inducing plasmids may beprepared using methods known to those skilled in the art. See Matzke andChilton, 1981; Leemans et al, 1981; Koekman et al, 1979.

FIG. 10 represents an octopine-type Ti plasmid with a T-DNA region 22which undergoes mutation to delete the tms and tmr genes and the rightborder. This results in a disarmed Ti plasmid with partial T-DNA region24. When plasmid 26 (such as pMON128) is inserted into a cell thatcarries the disarmed Ti plasmid 24, a crossover event occurs whichcreates a co-integrate Ti plasmid with disarmed T-DNA region 28. The LIHregion of homology is repeated in this Ti plasmid, but the disarmed Tiplasmid does not contain any oncogenic genes. Alternately, if only theright border had been deleted from T-DNA region 22, then the tms and tmrgenes and the octopine synthase (OCS) gene would be contained in theco-integrate disarmed Ti plasmid; however, they would have been locatedoutside of the T-DNA borders.

The disarmed co-integrate Ti plasmid is used to infect plant cells, andT-DNA region 28 enters the plant genome, as shown by transformed DNA 30.Plant cells transformed by disarmed T-DNA 28 have normal phytohormonemetabolism, and normal capability to be regenerated into differentiatedplants.

After pMON128 is inserted into A. tumefaciens cells, the desiredcrossover event will occur in a certain fraction of the cells. Cellswhich contain co-integrate plasmids (whether virulent or disarmed) maybe easily selected from other cells in which the crossover did notoccur, in the following manner. Plasmid pMON120 and its derivativescontain a marker gene (spc/str), which is expressed in A. tumefaciens.However, these plasmids do not replicate in A. tumefaciens. Therefore,the spc/str marker gene will not be replicated or stably inherited by A.tumefaciens unless the inserted plasmid combines with another plasmidthat can replicate in A. tumefaciens. The most probable suchcombination, due to the region of homology, is the co-integrate formedwith the Ti plasmid. A. tumefaciens cells which contain thisco-integrate plasmid can readily be identified and selected by growth ofthe cells on medium containing either spc or str, or both.

The Ti plasmid 28, shown in FIG. 10, contains two LIH regions. It ispossible that co-integrate Ti plasmids will undergo a subsequentcrossover event, wherein the two LIH regions will recombine. This eventis undesirable, since it can lead to a deletion of the DNA between theLIH regions, including the chimeric gene. However, this is not likely tolead to serious difficulties, for two reasons. First, this event islikely to occur at a relatively low probability, such as about 10⁻².Second, plasmid pMON120 and its derivatives, have been designed so thatthe selectable marker gene (spc/str) is located in the region of DNAthat would be deleted by the crossover event. Therefore, the selectiveconditions that are used to identify and culture Agrobacteria cellscontaining co-integrate plasmids will also serve to kill the descendantsof cells that undergo a subsequent crossover event which eliminates thechimeric gene from the Ti plasmid.

Only one of the LIH regions in the co-integrate Ti plasmid will beinserted into the plant genome, as shown in FIG. 10. This importantfeature results from the design of pMON120, and it distinguishes thisco-integrate plasmid from undesired co-integrate plasmids formed by theprior art. The LIH region which lies outside of the T-DNA borders willnot be inserted into the plant genome. This leads to at least twoimportant advantages. First, the presence of two LIH regions insertedinto the plant genome could result in crossover events which would leadto loss of the inserted genes in the transformed plant cells and theirprogeny. Second, the presence of two regions of DNA homology cansignificantly complicate efforts to analyze the DNA inserted into theplant genome (Matzke and Chilton, 1981).

After A. tumefaciens cells which contain the co-integrate Ti plasmidswith the chimeric genes have been identified and isolated, theco-integrate plasmids (or portions thereof) must be inserted into theplant cells. Eventually, methods may be developed to perform this stepdirectly. In the meantime, a method has been developed which may be usedconveniently and with good results. This method is described in twoseparate, simultaneously-filed applications, entitled “Transformation ofPlant Cells by Extended Bacterial Co-Cultivation” Ser. No. 458,413 and“Rapid Culture of Plant Protoplasts,” Ser. No. 538,412 The contents ofboth of those applications are hereby incorporated by reference. Themethod described in those applications may be briefly summarized asfollows.

The plant cells to be transformed are contacted with enzymes whichremove the cell walls. This converts the plant cells into protoplasts,which are viable cells surrounded by membranes. The enzymes are removed,and the protoplasts begin to regenerate cell wall material. At anappropriate time, the A. tumefaciens cells (which contain theco-integrate Ti plasmids with chimeric genes) are mixed with the plantprotoplasts. The cells are co-cultivated for a period of time whichallows the A. tumefaciens to infect the plant cells. After anappropriate co-cultivation period, the A. tumefaciens cells are killed,and the plant cells are propagated.

Plant cells which have been transformed (i.e., cells which have receivedDNA from the co-integrate Ti plasmids) and their descendants may beselected by a variety of methods, depending upon the type of gene(s)that were inserted into the plant genome. For example, certain genes maycause various antibiotics to be inactivated; such genes include thechimeric NOS-NPT II-NOS gene carried by pMON128. Such genes may serve asselectable markers; a group of, cells may be cultured on mediumcontaining the antibiotic which is inactivated by the chimeric geneproduct, and only those cells containing the selectable marker gene willsurvive.

A variety of genes may serve as scorable markers in plant cells. Forexample, pMON120 and its derivative plasmids, such as pMON128, carry anopaline synthase (NOS) gene which is expressed in plant cells. Thisgene codes for an enzyme which catalyzes the formation of nopaline.Nopaline is a non-detrimental compound which usually is accumulated atlow quantities in most types of plants; it can be easily detected byelectrophoretic or chromatographic methods.

If a plant is transformed by a gene which creates a polypeptide that isdifficult to detect, then the presence of a selectable marker gene (suchas the NOS-NPT II-NOS chimeric gene) or a scorable marker gene (such asthe NOS gene) in the transforming vector may assist in theidentification and isolation of transformed cells.

Virtually any desired gene may be inserted into pMON120 or other whichare designed to form co-integrates with plant tumor inducing or similarplasmids. For example, the Applicants have created a variety of chimericgenes, which are discussed in the previously-cited application,“Chimeric Genes Suitable for Expression in Plant Cells.”

The suitability of any gene for use in this invention may be determinedthrough routine experimentation by those skilled in the art. Such usageis not limited to chimeric genes; for example, this invention may beused to insert multiple copies of a natural gene into plant cells.

This invention is suitable for use with a wide variety of plants, as maybe determined through routine experimentation by those skilled in theart. For example, this invention is likely to be useful to transformcells from any type of plant which can be infected by bacteria from thegenus Agrobacterium. It is believed that virtually all dicotyledonousplants, and certain monocots, can be infected by one or more strains ofAgrobacterium. In addition, microorganisms of the genus Rhizobia arelikely to be useful for carrying co-integrate plasmids of thisinvention, as may be determined by those skilled in the art. Suchbacteria might be preferred for certain types of transformations orplant types.

Certain types of plant cells can be cultured in vitro and regeneratedinto differentiated plants using techniques known to those skilled inthe art. Such plant types include potatoes, tomatoes, carrots, alfalfaand sunflowers. Research in in vitro plant culture techniques isprogressing rapidly, and methods are likely to be developed to permitthe regeneration of a much wider range of plants from cells cultured invitro. Cells from any such plant with regenerative capacity are likelyto be transformable by the in vitro co-cultivation method discussedpreviously, as may be determined through routine experimentation bythose skilled in the art. Such transformed plant cells may beregenerated into differentiated plants using the procedures described inthe examples.

The in vitro co-cultivation method offers certain advantages in thetransformation of plants which are susceptible to in vitro culturing andregeneration. However, this invention is not limited to in vitro cellculture methods. For example, a variety of plant shoots and cuttings(including soybeans, carrots, and sunflowers) have been transformed bycontact with A. tumefaciens cells carrying the co-integrate plasmids ofthis invention. It is also possible to regenerate virtually any type ofplant from a cutting or shoot. Therefore, it may be possible to developmethods of transforming shoots or cuttings using virulent or preferablydisarmed co-integrate plasmids of this invention or mixtures thereof,and subsequently regenerating the transformed shoots or cuttings intodifferentiated plants which pass the inserted genes to their progeny.

As mentioned previously, it is not essential to this invention thatco-cultivation be utilized to transfer the co-integrate plasmids of thisinvention into plant cells. A variety of other methods are being used toinsert DNA into cells. Such methods include encapsulation of DNA inliposomes, complexing the DNA with chemicals such as polycationicsubstances or calcium phosphate, fusion of bacterial spheroplasts withplant protoplasts, microinjection of DNA into a cell, and induction ofDNA uptake by means of electric current. Although such methods have notbeen used to insert DNA into plant cells with satisfactory efficiency todate, they are being actively researched and they may be useful forinserting foreign genes into plant cells, using the intermediate vectorsand co-integrate plasmids of this invention, or plasmids derivedtherefrom.

This invention may be useful for a wide variety of purposes. Forexample, certain bacterial enzymes, such as 5-enol pyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) are inactivated bycertain herbicides; other enzymes, such as glutathione-S-transferase(GST), inactivate certain herbicides. It may be possible to insertchimeric genes into plants which will cause expression of such enzymesin the plants, thereby causing the plants to become resistant to one ormore herbicides. This would allow for the herbicide, which wouldnormally kill the untransformed plant, to be applied to a field oftransformed plants. The herbicide would serve as a weed-killer, leavingthe transformed plants undamaged.

Alternately, it may be possible to insert chimeric genes into plantswhich will cause the plants to create mammalian polypeptides, such asinsulin, interferon, growth hormone, etc. At an appropriate time, theplants (or cultured plant tissue) would be harvested. Using a variety ofprocesses which are known to those skilled in the art, the desiredprotein may be extracted from the harvested plant tissue.

An alternate use of this invention is to create plants with high contentof desired substances, such as storage proteins or other proteins. Forexample, a plant might contain one or more copies of a gene which codesfor a desirable protein. Additional copies of this gene may be insertedinto the plant by means of this invention. Alternately, the structuralsequence of the gene might be inserted into a chimeric gene under thecontrol of a different promoter which causes prolific transcription ofthe structural sequence.

The methods of this invention may be used to identify, isolate, andstudy DNA sequences to determine whether they are capable of promotingor otherwise regulating the expression of genes within plant cells. Forexample, the DNA from any type of cell can be fragmented, using partialendonuclease digestion or other methods. The DNA fragments can beinserted randomly into plasmids similar to pMON128. These plasmids,instead of having a full chimeric gene such as NOS-NPT II-NOS, will havea partial chimeric gene, with a cleavage site for the insertion of DNAfragments, rather than a NOS promoter or other promoter. The plasmidswith inserted DNA are then inserted into A. tumefaciens, where they canrecombine with the Ti plasmids. Cells having co-integrate plasmids areselected by means of the spc/str or other marker gene. The co-integrateplasmids are then inserted into the plant cells, by bacterialco-cultivation or other means. The plant cells will contain a selectablemarker structural sequence such as the NPT II structural sequence, butthis structural sequence will not be transcribed unless the inserted DNAfragment serves as a promoter for the structural sequence. The plantcells may be selected by growing them on medium containing kanamycin orother appropriate antibiotics.

Using this method, it is possible to evaluate the promoter regions ofbacteria, yeast, fungus, algae, other microorganisms, and animal cellsto determine whether they function as gene promoters in various types ofplant cells. It is also possible to evaluate promoters from one type ofplant in other types of plant cells. By using similar methods andvarying the cleavage site in the starting plasmid, it is possible toevaluate the performance of any DNA sequence as a 5′ non-translatedregion, a 3′ non-translated region, or any other type of regulatorysequence.

As used herein, “a piece of DNA” includes plasmids, phages, DNAfragments, and polynucleotides, whether natural or synthetic.

As used herein, a “chimeric piece of DNA” is limited to a piece of DNAwhich contains at least two portions (i.e., two nucleotide sequences)that were derived from different and distinct pieces of DNA. Forexample, a chimeric piece of DNA cannot be created by merely deletingone or more portions of a naturally existing plasmid. A chimeric pieceof DNA may be produced by a variety of methods, such as ligating twofragments from different plasmids together, or by synthesizing apolynucleotide wherein the sequence of bases was determined by analysisof the base sequences of two different plasmids.

As used herein, a chimeric piece of DNA is limited to DNA which has beenassembled, synthesized, or otherwise produced as a result of man-madeefforts, and any piece of DNA which is replicated or otherwise derivedtherefrom. “Man-made efforts” include enzymatic, cellular, and otherbiological processes, if such processes occur under conditions which arecaused, enhanced, or controlled by human effort or intervention; thisexcluses plasmids, phages, and polynucleotides which are created solelyby natural processes. As used herein, the term “derived from” shall beconstrued broadly. Whenever used in a claim, the term “chimeric” shallbe a material limitation.

As used herein, a “marker gene” is a gene which confers a phenotypicallyidentifiable trait upon the host cell which allows transformed hostcells to be distinguished from non-transformed cells. This includesscreenable, scorable, and selectable markers.

As used herein, a “region of homology” refers to a sequence of bases inone plasmid which has sufficient correlation with a sequence of bases ina different plasmid to cause recombination of the plasmid to occur at astatistically determinable frequency. Preferably, such recombinationshould occur at a frequency which allows for the convenient selection ofcells having combined plasmids, e.g., greater than 1 per 10⁶ cells. Thisterm is described more fully in a variety of publications, e.g., Leemanset al, 1981.

The term “chimeric gene” refers to a gene that contains at least twoportions that were derived from different and distinct genes. As usedherein, this term is limited to genes which have been assembled,synthesized, or otherwise produced as a result of man-made efforts, andany genes which are replicated or otherwise derived therefrom. “Man-madeefforts” include enzymatic, cellular, and other biological processes, ifsuch processes occur under conditions which are caused, enhanced, orcontrolled by human effort or intervention; this excludes genes whichare created solely by natural processes.

As used herein, a “gene” is limited to a segment of DNA which isnormally regarded as a gene by those skilled in the art. For example, aplasmid might contain a plant-derived promoter region and a heterologousstructural sequence, but unless those two segments are positioned withrespect to each other in the plasmid such that the promoter regioncauses the transcription of the structural sequence, then those twosegments would not be regarded as included in the same gene.

This invention relates to chimeric genes which have structural sequencesthat are “heterologous” with respect to their promoter regions. Thisincludes at least two types of chimeric genes:

1. DNA of a gene which is foreign to a plant cell. For example, if astructural sequence which codes for mammalian protein or bacterialprotein is coupled to a plant promoter region, such a gene would beregarded as heterologous.

2. A plant cell gene which is naturally promoted by a different plantpromoter region. For example, if a structural sequence which codes for aplant protein is normally controlled by a low-quantity promoter, thestructural sequence may be coupled with a prolific promoter. This mightcause a higher quantity of transcription of the structural sequence,thereby leading to plants with higher protein content. Such a structuralsequence would be regarded as heterologous with regard to the prolificpromoter.

However, it is not essential for this invention that the entirestructural sequence be heterologous with respect to the entire promoterregion. For example, a chimeric gene of this invention may be createdwhich would be translated into a “fusion protein”, i.e., a proteincomprising polypeptide portions derived from two separate structuralsequences. This may be accomplished by inserting all or part of aheterologous structural sequence into the structural sequence of a plantgene, somewhere after the start codon of the plant structural sequence.

As used herein, the phrase, “a promoter region derived from a specifiedgene” shall include a promoter region if one or more parts of thepromoter region were derived from the specified gene. For example, itmight be discovered that one or more portions of a particularplant-derived promoter region (such as intervening region 8, shown onFIG. 12) might be replaced by one or more sequences derived from adifferent gene, such as the gene that contains the heterologousstructural sequence, without reducing the expression of the resultingchimeric gene in a particular type of host cell. Such a chimeric genewould contain a plant-derived association region 2, intervening region4, and transcription initiation sequence 6, followed by heterologousintervening region 8, 5′ non-translated region 10 and structuralsequence 14. Such a chimeric gene is within the scope of this invention.

As used herein, the phrase “derived from” shall be construed broadly.For example, a structural sequence may be “derived from” a particulargene by a variety of processes, including the following:

1. The gene may be reproduced by various means such as inserting it intoa plasmid and replicating the plasmid by cell culturing, in vitroreplication, or other methods, and the desired sequence may be obtainedfrom the DNA copies by various means such as endonuclease digestion;

2. mRNA which was coded for by the gene may be obtained and processed invarious ways, such as preparing complementary DNA from the mRNA and thendigesting the cDNA with endonucleases;

3. The sequence of bases in the structural sequence may be determined byvarious methods, such as endonuclease mapping or the Maxam-Gilbertmethod. A strand of DNA which duplicates or approximates the desiredsequence may be created by various methods, such as chemical synthesisor ligation of oligonucleotide fragments.

4. A structural sequence of bases may be deduced by applying the geneticcode to the sequence of amino acid residues in a polypeptide.

Usually, a variety of DNA structural sequences may be determined for anypolypeptide, because of the redundancy of the genetic code. From thisvariety, a desired sequence of bases may be selected, and a strand ofDNA having the selected sequence may be created.

If desired, any DNA sequence may be modified by substituting certainbases for the existing bases. Such modifications may be performed for avariety of reasons. For example, one or more bases in a sequence may bereplaced by other bases in order to create or delete a cleavage site fora particular endonuclease. As another example, one or more bases in asequence may be replaced in order to reduce the occurrence of “stem andloop” structures in messenger RNA. Such modified sequences are withinthe scope of this invention.

A structural sequence may contain introns and exons; such a structuralsequence may be derived from DNA, or from an mRNA primary transcript.Alternately, a structural sequence may be derived from processed mRNA,from which one or more introns have been deleted.

The Applicants have deposited two cultures of E. coli cells containingplasmids pMON128 and pMON154 with the American Type Culture Collection(ATCC). These cells have been assigned ATCC accession numbers 39264 and39265, respectively.

The Applicants have deposited two cultures of E. coli cells containingplasmids pMON120 and pMON128 and a culture of A. tumefaciens containinga pMON128::Ti cointegrate plasmid, with the American Type CultureCollection (ATCC). These cells have been assigned ATCC accession numbers39263, 39264, and 39266, respectively. The Applicants have claimedcultures of microorganisms having the “relevant characteristics” ofthese cultures. As used herein, the “relevant characteristics” of a cellculture are limited to those characteristics which make the culturesuitable for a use which is disclosed, suggested or made possible by theinformation contained herein. Numerous characteristics of the culturemay be modified by techniques known to those skilled in the art; forexample, the cells may be made resistant to a particular antibiotic byinsertion of a particular plasmid or gene into the cells, or thespecified plasmids might be removed from the designated cells andinserted into a different strains of cells. Such variations are withinthe scope of this invention, even though they may amount to improvementsin the culture, which undoubtedly will occur after more researchers gainaccess to these cell cultures.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments of the invention discussed herein. Such equivalentsare within the scope of this invention.

EXAMPLE 1 Creation of Plasmid pMON41

A culture of E. coli, carrying a pBR325 plasmid (Bolivar, 1978) with theHindIII-23 fragment of pTiT37 (Hernalsteens, et al, 1980) inserted atthe HindIII site, was obtained from Drs. M. Bevan and M. D. Chilton,Washington University, St. Louis, Mo. Ten micrograms (ug) of the plasmidfrom this clone was digested with 10 units of HindIII (unless noted, allrestriction endonucleases used in these constructions were purchasedfrom New England Biolabs, Beverly, Mass. and used with buffers accordingto the supplier's instructions) for 1 hour at 37° C. The 3.4 kbHindIII-23 fragment was purified by adsorption on glass beads(Vogelstein and Gillespie, 1979) after separation from the other HindIIIfragments by electrophoresis on a 0.8% agarose gel. The purified 3.4 kbHindIII fragment (1.0 ug) was mixed with 1.0 ug of plasmid pBR327 DNA(Soberon, et al, 1980) that had been digested with both HindIII (2units, 1 hour, 37° C.) and calf alkaline phosphatase (CAP; 0.2 units, 1hour, 37° C.), de-proteinized with phenol, ethanol precipitated, andresuspended in 10 ul of TE (10 mM Tris HCl, pH8, 1 mM EDTA). One unit ofT4 DNA ligase (prepared by the method of Murray et al, 1979) was addedto the fragment mixture. One unit is defined as the concentrationsufficient to obtain greater than 90% circularization of one microgramof HindIII linearized pBR327 plasmid in 5 minutes at 22° C. The mixedfragments were contained in a total volume of 15 ul of 25 mM Tris-HClpH8, 10 mM MgCl₂, 1 mM dithiotheitol, 200 uM spermidine HCl and 0.75 mMATP (ligase buffer).

The mixture was incubated at 22° for 3 hours and then mixed with E. coliC600 recA56 cells that were prepared for transformation by treatmentwith CaCl₂(Maniatis et al, 1982). Following a period for expression ofthe ampicillin resistance determinant carried by the pBR327 vector,cells were spread on LB solid medium plates (Miller, 1972) containingampicillin at 200 ug/ml. After incubation at 37° C. for 16 hours,several hundred clones were obtained. Plasmid mini-preps (Ish-Horowiczand Burke, 1981) were performed on 24 of these colonies and aliquots ofthe plasmid DNA's obtained (0.1 ug) were digested with HindIII todemonstrate the presence of the 3.4 kb HindIII fragment. One plasmiddemonstrated the expected structure and was designated pMON38. pMON38DNA were prepared by Triton X-100 lysis and CsC1 gradient procedure(Davis et al, 1980).

Fifty ug of pMON38 DNA were digested with HindIII and BamHI (50 unitseach, 2 hours, 37°) and the 2.3 kb HindIII-BamHI fragment was purifiedas described above. The purified fragment (1 ug) was mixed with 1 ug ofthe 2.9 kb HindIII-BamHI fragment of the pBR327 vector purified asdescribed above. Following ligation (T4 DNA ligase, 2 units) andtransformation of E. coli cells as described above, fiftyampicillin-resistant colonies were obtained. DNAs from twelve plasmidmini-preps were digested with HindIII and BamHI to ascertain thepresence of the 2.3 kb fragment. One plasmid of the correct structurewas chosen and designated pMON41, as shown in FIG. 2. A quantity of thisDNA was prepared as described above.

EXAMPLE 2 Creation of M13 Clone M-4

Thirty ug of plasmid pMON38 (described in Example 1) were digested withRsaI (30 units, 2 hours, 37° C.) and the 1100 bp RsaI fragment waspurified after separation by agarose gel electrophoresis using the glassbead method described in the previous example. The purified 1100 bpRsaI-RsaI fragment (1 ug) was digested with 2 units of BamHI and theBamHI was inactivated by heating. This DNA was mixed with 0.2 ug ofphage M13 mp8RF DNA which had been previously digested with SmaI andBamHI (2 units each, 1 hour, 37°) and 0.2 units of calf alkalinephosphotase (CAP). Following ligation with 100 units of T4 DNA ligase,transformation of E. coli JM101 cells as described in the previousexample, the transformed cells were mixed with soft agar and platedunder conditions that allow the identification of recombinant phage(Messing and Vieira, 1982). Twelve recombinant phage producing cellswere picked and RF plasmid mini-preps were obtained as described in theprevious example. The RF DNAs were digested with BamHI and SmaI to provethe presence of the 720 bp RsaI-BamHI fragment. One of the recombinantRF DNAs carrying the correct fragment was designated M13 mp8 M-4. Thisprocedure is represented in FIG. 3. M-4 RF DNA was prepared using theprocedures of Ish-Horowicz and Burke, 1981 and Colman et al, 1978.

EXAMPLE 3 Construction of pMON109

Twenty ug of plasmid pGV3106 (Hernalsteens et al 1980, prepared by themethod of Currier and Nester 1976) was digested with HindIII (20 units,2 hours, 37°) and mixed with 2 ug of HindIII-digested pBR327. Followingligation (T4 DNA ligase, 2 units) and transformation of E. coli cells asdescribed above, one colony resistant to trimethoprim (100 ug/ml) andampicillin was obtained. Digestion of plasmid DNA from this celldemonstrated the presence of a 6 kb HindIII fragment. This plasmid wasdesignated pMON31.

Plasmid pMON31 from a mini-prep (0.5 ug) was digested with EcoRI (1unit, 1 hour, 37° C.) and the endonuclease was inactivated by heating(10 min, 70° C.). The 8.5 kb plasmid fragment was re-circularized in aligation reaction of 100 ul (T4 DNA ligase, 1 unit) and used totransform E. coli cells with selection for ampicillin and streptomycin(25 ug/ml) resistant colonies. Plasmid mini-prep DNA's from six cloneswere digested with EcoRI to ascertain loss of the 850 by fragment. Oneplasmid lacking the 850 bp EcoRI fragment was designated pMON53. Thisplasmid was introduced into E. coli GM42 dam⁻ cells (Bale et al, 1979)by transformation as described.

Plasmid pMON53 (0.5 ug) from a mini-prep prepared from dam cells wasdigested with ClaI, and recircularized in dilute solution as describedabove. Following transformation of E. coli GM42 cells and selection forampicillin and spectinomycin (50 ug/ml) resistant clones, fifty colonieswere obtained. Digestion of plasmid mini-prep DNA's from six coloniesshowed that all lacked the 2 kb ClaI fragment. One of these plasmids wasdesignated pMON54, as represented in FIG. 4. Plasmid DNA was prepared asdescribed in Example 1.

Plasmid pMON54 DNA (20 ug) was digested with EcoRI and PstI (20 units ofeach, 2 hours, 37° C.) and the 5.7 kb fragment was purified from agarosegels using NA-45 membrane (Schleicher and Schuell, Keene, N. H.).

The purified 5.7 kb fragment (0.5 ug) was mixed with 0.3 ug of a 740 bpEcoRI-PstI fragment obtained from M13 mp8 M-4 RF DNA (described inExample 2) which was purified using NA-45 membrane. Following ligation(T4 DNA ligase, 2 units), transformation of E. coli GM42 dam⁻ cells, andselection for spectinomycin resistant cells, twenty colonies wereobtained. Plasmid mini-prep DNA's prepared from twelve of these cloneswere digested with PstI and EcoRI to demonstrate the presence of the 740bp fragment. One plasmid carrying this fragment was designated pMON64.quantity of this plasmid DNA was prepared as described in Example 1.

DNA (0.5 ug) of pMON64 was digested with ClaI (1 unit, 1 hour, 37° C.),the ClaI was heat inactivated, and the fragments rejoined with T4 DNAligase (1 unit). Following transformation and selection forspectinomycin resistant cells, plasmid mini-preps from twelve colonieswere made. The DNA's were digested with BamHI and EcoRI to determine theorientation of the 2 kb ClaI fragment. Half of the clones contained theClaI fragment in the inverse orientation of that in pMON64. One of theseplasmids was designated pMON109, as represented in FIG. 5. DNA wasprepared as described in Example 1.

EXAMPLE 4 Creation of Plasmid pMON113

Plasmid pNW31C-8,29C (Thomashow et al, 1980) was obtained from Dr. S.Gelvin of Purdue University, West Lafayette, I N. This plasmid carriesthe pTiA6 7.5 kb Bam-8 fragment. The Bam-8 fragment was purified from 50ug of BamHI-digested pNW31C-8,29C using NA-45 membrane as described inprevious examples. The purified 7.5 kb Bam-8 fragment (1.0 ug) was mixedwith 0.5 ug of pBR327 vector DNA which had been previously digested withboth BamHI (2 units) and 0.2 units of calf alkaline phosphatase (CAP)for 1 hour at 37°; the mixture was deproteinized and resuspended asdescribed in previous examples. The mixed fragments were treated with T4ligase (2 units), used to transform E. coli C600 recA cells andampicillin-resistant colonies were selected as described previously.Mini-preps to obtain plasmid DNA were performed on twelve of theseclones. The DNA was digested with BamHI to demonstrate the presence ofthe pBR327 vector and 7.5 kb Bam-8 fragments. One plasmid demonstratingboth fragments was designated pMON90. DNA was prepared as described inExample 1.

Twenty-five ug of pMON90 DNA were digested with BglII (25 units, 2hours, 37°) and the 2.6 kb BglII fragment was purified using NA-45membrane. To create blunt ends, the fragment (2 ug) was resuspended in10 ul of 50 mM NaCl, 6.6 mM Tris-HCl pH8, 6.6 mM MgCl₂ and 0.5 mMdithiothreitol (Klenow Buffer). The 4 deoxy-nucleoside triphosphates(dATP, dTTP, dCTP, and dGTP) were added to a final concentration of 1 mMand one unit of E. coli large Klenow fragment of DNA polymerase I (NewEngland Biolabs, Beverly, Mass.) was added. After incubation for 20minutes at 22° C., the Klenow polymerase was heat inactivated and 10units of HindIII was added. The HindIII digestion was carried out for 1hour at 37° and then the enzyme was heat inactivated. The HindIII-BglII(blunt) fragments (1 ug) were added to 0.25 ug of the 2.2 kbHindIII-PvuII fragment of pBR322 (Bolivar, et al, 1977) which had beengenerated by HindIII and PvuII digestion then treating with calfalkaline phosphatase as described in previous examples. After ligationusing 100 units of T4 DNA ligase, transformation of E. coli LE392 cellsand selection of ampicillin-resistant colonies as described in previousexamples, nineteen colonies were obtained. Plasmid mini-preps wereprepared from twelve colonies and digested with HindIII to determine thesize of the recombinant plasmid and with Sinai to determine that thecorrect fragment had been inserted. One plasmid with the correctstructure was designated pMON113, as shown in FIG. 6. Plasmid DNA wasprepared as described in Example 1.

EXAMPLE 5 Creation of Plasmid pMON120

Twenty ug of plasmid pMON109 (described in Example 3) were digested withEcoRI and BamHI (20 units each, 2 hours, 37° C.) and the 3.4 kbBamHI-EcoRI fragment was purified using NA-45 membrane as described inprevious examples. Twenty ug of plasmid pMON41 (described in Example 1)were digested with BamHI and PvuI (20 units each, 2 hours, 37° C.) andthe 1.5 kb BamHI-PvuI fragment purified using NA-45 membrane asdescribed in previous examples.

Twenty ug of pMON113 DNA (described in Example 4) were digested withPvuI and EcoRI (2 units each, 2 hr, 37° C.) and the 3.1 kb PvuI-EcoRIfragment was purified using NA-45 membrane as above. To assemble plasmidpMON120, the 3.1 kb EcoRI-PvuI pMON113 fragment (1.5 ug) was mixed with1.5 ug of the 3.4 kb EcoRI-BamHI fragment from pMON109. After treatmentwith T4 ligase (3 units) for 16 hours at 10° C., the ligase wasinactivated by heating (10 minutes, 70° C.), and 5 units of BamHI wasadded. Digestion continued for 30 minutes at 37° at which time the BamHIendonuclease was inactivated by heating as above. Next, 0.75 ug of the1.5 kb PvuI-BamHI fragment from pMON41 was added along with T4 DNAligase (2 units) and fresh ATP to 0.75 mM final concentration. The finalligase reaction was carried out for 4 hours at 22° C. at which time themixture was used to transform E. coli LE 392 cells with subsequentselection for spectinomycin resistant cells as described previously.Plasmid mini-preps from twelve out of several thousand colonies werescreened for plasmids of approximately 8 kb in size containing singlesites for BamHI and EcoRI. One plasmid showing the correct structure wasdesignated pMON120, which is shown in FIG. 7 with an alternate method ofconstruction. pMON120 DNA was prepared as described in Example 1.

A culture of E. coli containing pMON120 has been deposited with theAmerican Type Culture Collection. This culture has been assignedaccession number 39263.

EXAMPLE 6 Creation of Plasmids pMON128 and pMON129

Plasmid pMON75 (described in detail in a separate application entitled“Chimeric Genes Suitable for Expression in Plant Cells,” previouslycited and incorporated contains a chimeric NOS-NPT II-NOS gene. Thisplasmid (and pMON128, described below) may be digested by EcoRI and a1.5 kb fragment may be purified which contains the NOS-NPT II-NOS gene.

Plasmid pMON120 was digested with EcoRI and treated with calf alkalinephosphatase. After phenol deproteinization and ethanol precipitation,the EcoRI-cleaved pMON120 linear DNA was mixed with 0.5 ug of the 1.5 kbEcoRI chimeric gene fragment from pMON75 or 76. The mixture was treatedwith 2 units of T4 DNA ligase for 1 hour at 22°. After transformation ofE. coli cells and selection of colonies resistant to spectinomycin (50ug/ml), several thousand colonies appeared. Six of these were picked,grown, and plasmid mini-preps made. The plasmid DNA's were digested withEcoRI to check for the 1.5 kb chimeric gene insert and with BamHI todetermine the orientation of the insert. BamHI digestion showned that inpMON128 the chimeric gene was transcribed in the same direction as theintact nopaline synthase gene of pMON120. A culture of E. colicontaining pMON128 has been deposited with the American Type CultureCollection. This culture has been assigned accession number 39264. Theorientation of the insert in pMON129 was opposite that in pMON128; theappearance of an additional 1.5 kb BamHI fragment in digests of pMON129showed that plasmid pMON129 carried a tandem duplication of the chimericNOS-NPT II-NOS gene, as shown in FIG. 8.

EXAMPLE 7 Creation of Co-integrate Plasmid pMON128::pTiB6S3Tra^(C)

Plasmid pMON128 (described in Example 6) was transferred to achloramphenicol resistant Agrobacterium tumefaciens strain GV3111=C58C1carrying Ti plasmid pTiB6S3tra^(C) (Leemans, et al, 1982) using atri-parental plate mating procedure, as follows. 0.2 ml of a culture ofE. coli carrying pMON128 was mixed with 0.2 ml of a culture of E. colistrain HB101 carrying a pRK2013 plasmid (Ditta, et al, 1980) and 0.2 mlof GV3111 cells. The mixture of cells was cultured in Luria Broth (LB),spread on an LB plate, and incubated for 16 to 24 hours at 30° C. toallow plasmid transfer and generation of co-integrate plasmids. Thecells were resuspended in 3 ml of 10 mM MgSO₄ and 0.2 ml aliquot wasthen spread on an LB plate containing 25 ug/ml chloramphenicol and 100ug/ml each of spectinomycin and streptomycin. After incubation for 48 hrat 30°, approximately 10 colonies were obtained. One colony was chosenand grow at 30° C. in LB medium containing chloramphenicol,spectinomycin, and streptomycin at the concentrations given above.

A separate type of co-integrate plasmid for use in control experimentswas prepared by inserting pMON120 into A. tumefaciens cells, andselecting for cells with co-integrate plasmids using spectinomycin andstreptomycin, as described above. Like pMON120, these plasmids do notcontain the chimeric NOS-NPT II-NOS gene.

EXAMPLE 8 Solutions Used in Plant Cell Cultures

The following solutions were used by the

Applicants:

 per liter Enzyme mix: Cellulysin 5 g Macerozyme 0.7 g Ampicillin 0.4 gKH₂PO₄ 27.2 mg KNO₃ 101 mg CaCl₂ 1.48 g MgSO₄•7H₂O 246 mg KI 0.16 mgCuSO₄•5H₂O 0.025 mg Mannitol 110 g MS9: MS salts(see below) 4.3 gSucrose 30.0 g B5 vitamins (see below) 1 ml Mannitol 90.0 gPhytohormones: Benzyladenine (BA) 0.5 mg 2,4-D 1 mg MS-ES MS salts 4.3 gSucrose 30 g B5 Vitamins 1 ml Mannitol 30 g Carbenicillin 10 mgPhytohormones: Indole acetic acid 0.1 mg MSO: MS salts 4.3 g Sucrose30.0 g B5 vitamins 1 ml Feeder plate MS salts 4.3 g medium: Sucrose 30.0g B5 vitamins 1 ml Mannitol 30.0 g Phytohormones: BA 0.5 mg Ms2C MSsalts 4.3 g Sucrose 30 g B5 vitamins 1 ml Phytohormones:chlorophenoxyacetic 2 mg acid MS104: MS salts 4.3 g Sucrose 30.0 g B5vitamins 1 ml Phytohormones: BA 0.1 mg NAA 1 mg MS11: MS salts 4.3 gSucrose 30.0 g B5 vitamins 1 ml Phytohormones: Zeatin 1 mg B5 Vitaminmyo-inositol 100 g stock: thiamine HCl 10 g nicotinic acid 1 gpyrodoxine HCl 1 g Float rinse: MS salts 0.43 g Sucrose 171.2 g PVP-4040.0 gMS salts are purchased pre-mixed as a dry powder from GibcoLaboratories, Grand Island, N.Y.

EXAMPLE 9 Preparation of Protoplasts

Mitchell petunia plants were grown in growth chambers with two or threebanks of fluorescent lamps and two banks of incandescent bulbs (about5,000 lux). The temperature was maintained at a constant 21° C. and thelights were on for 12 hours per day. Plants were grown in a 50/50 mix ofVermiculite and Pro-mix BX (Premier Brands Inc., Canada). Plants werewatered once a day with Hoagland's nutrient solution. Tissue was takenfrom dark green plants with compact, bushy growth. Leaves weresterilized in a solution of 10% commercial bleach and a small amount ofdetergent or Tween 20 for 20 minutes with occasional agitation. Leaveswere rinsed two or three times with sterile distilled water, Thin strips(about 1 mm) were cut from the leaves, perpendicular to the main rib.The strips were placed in the enzyme mix at a ratio of about 1 g tissueto 10 ml enzymes. The dishes were sealed with parafilm, and incubated inthe dark or under low, indirect light while gently agitatingcontinuously (e.g., 40 rpm on gyrotary shaker). Enzymic incubationsgenerally were run overnight, about 16-20 hours.

The digestion mixture was sieved through a 68, 74, or 88 um screen toremove large debris and leaf material. The filtrate was spun at 70-100 gfor five minutes to pellet the protoplasts. The supernatant was decantedand the pellet was gently resuspended in float rinse solution. Thissuspension was poured into babcock bottles. The bottles were filled to 2or 3 cm above the base of the neck. 1 ml of growth medium MS9 wascarefully layered on top of the float rinse.

The Babcock bottles were balanced and centrifuged at 500 to 1000 rpm for10 to 20 minutes. The protoplasts formed a compact band in the neck atthe interface. The band was removed with a pipette, taking care not topick up any excess float rinse. The protoplasts were diluted into MS9.At this point, the protoplasts were washed with MS9 or diluted forplating without washing.

Protoplasts were suspended in MS9 medium at 5×10⁴per ml, and plated intoT-75 flasks, at 6 ml per flask. Flasks were incubated on a level surfacewith dim, indirect light or in the dark at 26-28° C. On the third dayfollowing the removal of the enzymes from the leaf tissue, MSO (mediumwhich does not contain mannitol) was added to each flask, using anamount equal to one-half the original volume. The same amount of MSO isadded again on day 4. This reduces the mannitol concentration to about0.33 M after the first dilution, and about 0.25 M after the seconddilution.

EXAMPLE 10 Co-Cultivation with Bacteria

On day 5 following protoplast isolation, five to seven day old tobaccosuspension cultures (TXD cells) were diluted (if necessary) with MS2Cmedium to the point where 1 ml would spread easily over the surface ofagar medium in a 100×15 mm petri plate [this is a 10 to 15% suspension(w/v)]. The agar medium was obtained by mixing 0.8% agar with MS-ESmedium, autoclaving the mixture, and cooling the mixture until itsolidifies in the plate. One ml of the TXD suspension was spread over 25ml of feeder plate medium. An 8.5 cm disc of Whatman #1 filter paper waslaid over the TXD feeder cells and smoothed out. A 7 cm disc of the samepaper was placed in the center of the larger one.

Separately, aliquots of a culture of A. tumefaciens cells (grown inyeast extract peptone medium) were added to the flasks which containedthe plant cells. One set of aliquots contained cells with thepMON128::Ti co-integrate plasmids having chimeric NOS-NPT II-NOS genes.The other set of aliquots contained cells with the pMON120::Tico-integrate plasmids, which do not have chimeric NOS-NPT II-NOS genes.

The bacteria were added to the flasks to a density of 10⁸ cells/ml. 0.5ml of the cell mixture was spread in a thin layer on the surface of the7 cm filter paper disc. The plates were wrapped in parafilm or plasticbags and incubated under direct fluorescent lighting, no more than fiveplates in a stack.

Within seven days, colonies were discernable. Within 14 days, the 7 cmdiscs, with colonies adhering to them, were transferred to new MSO agarmedium (without feeder cells)_containing 500 ug/ml carbenicillin, aswell as 50 ug/ml of kanamycin sulfate (Sigma, St. Louis, Mo.). Withintwo weeks, vigorously growing green colonies could be observed on theplates which contained plant cells that had been co-cultured with A.tumefaciens strains containing the pMON128 co-integrate NOS-NPT IIplasmid. No transformed colonies were detected on plates which containedplant cells that had been co-cultured with A. tumefaciens strainscontaining the pMON120 co-integrate plasmid. The kanamycin resistanttransformants are capable of sustained growth in culture mediumcontaining kanamycin. Southern blotting experiments (as described in E.Southern, J. Mol. Biol. 98: 503 (1975) confirmed that these cellscontain the chimeric NOS-NPT II gene.

Both sets of transformed cells (and a third set of cells which had beentransformed in the same manner by a chimeric gene coding for the enzymeNPT type I) were assayed for resistance to kanamycin. The results areindicated in FIG. 11.

EXAMPLE 11 Regeneration of Transformed Plants

The transformed kanamycin-resistant colonies described in Example 10contained both tumorous and non-tumorous cells, as described in FIG. 9and the related text. The following procedure was used to isolatenon-tumorous transformed cells from tumorous transformed cells, and toregenerate differentiated plant tissue from the non-tumorous cells.

Colonies were grown on MS104 agar medium containing 30 ug/ml kanamycinsulfate and 500 ug/ml carbenicillin until they reached about 1 cm indiameter. Predominantly tumorous colonies appear a somewhat paler shadeof green and are more loosely organized than predominantly non-tumorouscolonies. Predominantly non-tumorous colonies were removed from theMS104 medium and placed upon MS11 medium containing 30 ug/ml kanamycinand 500 ug/ml carbenicillin. As the colonies continued to grow, coloniesthat appeared pale green and loosely organized were removed anddiscarded.

MS11 medium contains zeatin, a phytohormone which induces shootingformation in non-tumorous colonies. Several shoots were eventuallyobserved sprouting from kanamycin-resistant colonies. These shoots maybe grown to a desired size, cut off by a sharp blade, and inserted intoagar medium without phytohormones, such as MSO, where they may generateroots. If desired, the medium may be supplemented by napthalene aceticacid to induce shooting. The shoots may be grown to a desired size inthe agar medium, and then transferred into soil. If properly cultivated,such plants will grow to maturity and generate seed. The acquired traitwill be inherited by progeny according to classic Mendelian genetics.

EXAMPLE 12 Creation Of pMON1001

Fifty micrograms (ug) of lambda phage bbkan-1 DNA (Berg et al, 1975)were digested with 100 units of HindIII (all restriction endonucleaseswere obtained from New England Biolabs, Beverly, Mass., and were usedwith buffers according to the suppliers instructions, unless otherwisespecified) for 2 hours at 37° C. After heat-inactivation (70° C., 10minutes), the 3.3 kb Tn5 HindIII fragment was purified on a sucrosegradient. One ug of the purified HindIII fragment was digested withBamHI (2 units, 1 hr, 37° C.), to create a 1.8 kb fragment. Theendonuclease was heat inactivated.

Plasmid pBR327 (Soberon et al, 1981), 1 ug, was digested with HindIIIand BamHI (2 units each, 2 hours, 37° C.) Following digestion, theendonucleases were heat inactivated and the cleaved pBR327DNA was addedto the BamHI-HindIII Tn5 fragments. After addition of ATP to aconcentration of 0.75 mM, 10 units of T4 DNA ligase (prepared by themethod of Murray et al, 1979) was added, and the reaction was allowed tocontinue for 16 hours at 12°-14° C. One unit of T4 DNA ligase will give90% circularization of one ug of HindIII-cleaved pBR327 plasmid in 5minutes at 22° C.

The ligated DNA was used to transform CaCl₂-shocked E. coli C600 recA56cells (Maniatis et al, 1982). After expression in Luria broth (LB) for 1hour at 37° the cells were spread on solid LB media plates containing200 ug/ml ampicillin and 40 ug/ml kanamycin. Following 16 hoursincubation at 37° C., several hundred colonies appeared. Plasmidmini-prep DNA was prepared from six of these. (Ish-Horowicz and Burke,1981). Endonuclease digestion showed that all six of the plasmidscarried the 1.8 kb HindIII-BamHI fragment. One of those isolates wasdesignated as pMON1001 as shown in FIG. 17.

EXAMPLE 13 Creation of pMON40

-   -   Five ug of plasmid pMON1001 (described in Example 12) was        digested with SmaI. The reaction was terminated by phenol        extraction, and the DNA was precipitated by ethanol. A BamHI        linker CCGGATCCGG (0.1 ug), which had been phosphorylated with        ATP and T4 polynucleotide kinase (Bethesda Research Laboratory,        Rockville, Md.) was added to 1 ug of the pMON 1001 fragment. The        mixture was treated with T4 DNA ligase (100 units) for 18 hours        at 14° C. After heating at 70° C. for 10 minutes to inactivate        the DNA ligase, the DNA mixture was digested with BamHI        endonuclease (20 units, 3 hours, 37° C.) and separated by        electrophoresis on an 0.5% agarose gel. The band corresponding        to the 4.2 kb SmaI-BamHI vector fragment was excised from the        gel. The 4.2 kb fragment was purified by absorption on glass        beads (Vogelstein and Gillespie, 1979), ethanol precipitated and        resuspended in 20 ul of DNA ligase buffer with ATP. T4 DNA        ligase (20 units) was added and the mixture was incubated for        1.5 hours at room temperature. The DNA was mixed with rubidium        chloride-shocked in E. coli C600 cells for DNA transformation.        (Maniatis et al, 1982). After expression for 1 hour at 37° C. in        LB, the cells were spread on LB plates containing 200 ug/ml of        ampicillin and 20 ug/ml kanamycin. The plates were incubated at        37° C. for 16 hours. Twelve ampicillin-resistant,        kanamycin-resistant colonies were chosen, 2 ml cultures were        grown, and mini-plasmid preparations were performed.        Endonuclease mapping of the plasmids revealed that ten of the        twelve contained no SmaI site and a single BamHI site, and were        of the appropriate size, 4.2 kb. The plasmid from one of the ten        colonies was designated as pMON40, as shown in FIG. 17.

EXAMPLE 14 Creation of NOS Promoter Fragment

An oligonucleotide with the following sequence, 5′-TGCAGATTATTTGG-3′,was synthesized (Beaucage and Carruthers, 1981, as modified by Adams etal, 1982). This oligonucleotide contained a ³²P radioactive label, whichwas added to the 5′ thymidine residue by polynucleotide kinase.

An M13 mp 7 derivative, designated as S1A, was given to Applicants by M.Bevan and M. D. Chilton, Washington University, St. Louis, Mo. To thebest of Applicants' knowledge and belief, the S1A DNA was obtained bythe following method. A pTiT37 plasmid was digested with HindIII, and a3.4 kb fragment was isolated and designated as the HindIII-23 fragment.This fragment was digested with Sau3a, to create a 344 bp fragment withSau3a ends. This fragment was inserted into double-stranded, replicativeform DNA from the M13 mp 7 phage vector (Messing et al, 1981) which hadbeen cut with BamHI. Two recombinant phages with 344 bp insertsresulted, one of which contained the anti-sense strand of the NOSpromoter fragment. That recombinant phage was designated as S1A, and aclonal copy was given to the Applicants.

The Applicants prepared the single-stranded form of the S1A DNA (14.4ug; 6 pmol), and annealed it (10 minutes at 70° C., then cooled to roomtemperature) with 20 pmol of the 14-mer oligonucleotide, mentionedabove. The oligonucleotide annealed to the Sau3a insert at bases 286-300as shown on FIGS. 15 and 16.

200 ul of the S1A template and annealed oligonucleotide were mixed withthe four dNTP's (present at a final concentration of 1 mM, 25 ul) and 50ul of Klenow polymerase. The mixture incubated for 30 minutes at roomtemperature. During this period, the polymerase added dNTP's to the 3′end of the oligonucleogide. The polymerase was heat-inactivated (70° C.,3 minutes), and HaeIII (160 units) were added. The mixture was incubated(1 hour, 55° C.), the HaeIII was inactivated (70° C., 3 minutes), andthe four dNTP's (1 mM, 12 ul) and T4 DNA polymerase (50 units) wereadded. The mixture was incubated (1 hour, 37° C.) and the polymerase wasinactivated (70° C., 3 minutes). This yielded a fragment of about 570bp. EcoRI (150 units) was added, the mixture was incubated (1 hour, 37°C.) and the EcoRI was inactivated (70° C., 3 minutes).

Aliquots of the mixture were separated on 6% acrylamide with 25%glycerol. Autoradiography revealed a radioactively labelled band about310 bp in size. This band was excised. The foregoing procedure isindicated by FIG. 16.

EXAMPLE 15 Creation of pMON58

Five ug of plasmid pMON40 (described in Example 13) were digested withBglII (10 units, 1.5 hour, 37° C.), and the BglII was inactivated (70°C., 10 minutes). The four dNTP's (1 mM, 5 ul) and Klenow polymerase (8units) were added, the mixture was incubated (37° C., 40 minutes), andthe polymerase was inactivated (70° C., 10 minutes). EcoRI (10 units)was added and incubated (1 hour, 37° C.), and calf alkaline phosphatase(CAP) was added and incubated (1 hour, 37° C.). A fragment of about 3.9kb was purified on agarose gel using NA-45 membrane (Scheicher andScheull, Keene N H). The fragment (1.0 pM) was mixed with the NOSpromoter fragment (0.1 pM), described in Example 3, and with T4 DNAligase (100 units). The mixture was incubated (4° C., 16 hours). Theresulting plasmids were inserted into E. coli cells, which were selectedon media containing 200 ug/ml ampicillin. Thirty-six clonal Amp^(R)colonies were selected, and mini-preps of plasmids were made from thosecolonies. The plasmid from one colony demonstrated a 308 by EcoRI-BglIIfragment, a new SstII cleavage site carried by the 308 bp NOS fragment,and a new PstI site. This plasmid was designated as pMON58, as shown inFIG. 18. pMON58 DNA was prepared as described above.

EXAMPLE 16 Creation of pMON42

-   -   Plasmid pBR325-HindIII-23, a derivative of plasmid pBR325        (Bolivar, 1978) carrying the HindIII-23 fragment of pTIT37 (see        FIG. 14) in the HindIII site, was given to Applicants by M.        Bevan and M. D. Chilton, Washington University, St. Louis, Mo.        DNA of this plasmid was prepared and 30 ug were digested with        HindIII (50 units) and BamHI (50 units). The 1.1 kb        HindIII-BamHI fragment was purified by adsorption on glass beads        (Vogelstein and Gillespie, 1979) after agarose gel        electrophoresis. The purified fragment (0.5 ug) was added to 0.5        ug of the 2.9 kb HindIII-BamHI fragment of pBR327. After        treatment with DNA ligase (20 units, 4 hours, 22° C.), the        resulting plasmids were introduced to E. coli C600 cells. Clones        resistant to ampicillin at 200 ug/ml were selected on solid        media; 220 clones were obtained. Minipreps of plasmid DNA were        made from six of these clones and tested with the presence of a        1.1 kb fragment after digestion with HindIII and BamHI. One        plasmid which demonstrated the correct insert was designated        pMON42. Plasmid pMON42 DNA was prepared as described in previous        examples.

EXAMPLE 17 Creation of M13 Clone M-2

Seventy-five ug of plasmid pMON42 (described in Example 16) preparedfrom dam—E. coli cells were digested with RsaI and BamHI (50 units ofeach, 3 hours, 37° C.) and the 720 bp RsaI-BamHI fragment was purifiedusing NA-45 membrane. Eight ug of the purified 720 bp BamHI-RsaIfragment were digested with MboI (10 minutes, 70° C.), the ends weremade blunt by filling in with the large Klenow fragment of DNApolymerase I and the-four dNTP's. Then 0.1 ug of the resulting DNAmixture was added to 0.05 ug of M13 mp 8 previously digested with SmaI(1 unit, 1 hour 37° C.) and calf alkaline phosphatase (0.2 units). Afterligation (10 units of T4 DNA ligase, 16 hours, 12° C.) and transfectionof E. coli JM101 cells, several hundred recombinant phage were obtained.Duplex RF DNA was prepared from twelve recombinant, phage-carryingclones. The RF DNA (0.1 ug) was cleaved with EcoRI, (1 unit, 1 hour, 37°C.), end-labeled with ³²P-dATP and Klenow polymerase, and re-digestedwith BamHI (1 unit, 1 hour, 37° C.). The EcoRI and BainHI sites span theSmaI site. Therefore, clones containing the 260 bp MboI fragment couldbe identified as yielding a labelled 270 bp fragment afterelectrophoresis on 6% poly-acrylamide gels and autoradiography. Four ofthe twelve clones carried this fragment. The orientation of the insertwas determined by digestion of the EcoRI-cleaved, end-labeled RF DNA(0.1 ug) with HinfI (1 unit, 1 hour, 37° C.). HinfI cleaves the 260 byMboI fragment once 99 bp from the 3′ end of the fragment and again 42 bpfrom the end nearest the NOS coding region. Two clones of eachorientation were obtained. One clone, digested as M-2 as shown in FIG.19, contained the 260 bp fragment with the EcoRI site at the 3′ end ofthe fragment. M-2 RF DNA was prepared using the procedures of Messing,et al 1981.

EXAMPLE 18 Creation of pMON75 and pMON76

Fifty ug of M-2 RF DNA (described in Example 17) were digested with 50units of EcoRI and 50 units of BamHI for 2 hours at 37°. The 270 bpfragment (1 ug) was purified using agarose gel and NA-45 membrane.Plasmid pMON58 (described in Example 4) was digested with EcoRI andBamHI (50 ug, 50 units each, 2 hours, 37° C.) and the 1300 bp fragmentwas purified using NA-45 membrane. The 270 bp EcoRI-BamHI (0.1 ug) and1300 bp EcoRI-BamHI (0.5 ug) fragments were mixed, treated with T4 DNAligase (2 units) for 12 hours at 14° C. After heating at 70° C. for 10minutes to inactivate the ligase, the mixture was treated with EcoRI (10units) for 1 hour at 37° C., then heated to 70° C. for 10 minutes toinactivate the EcoRI. This completed the assembly of a chimericNOS-NPTII-NOS gene on a 1.6 kb fragment, as shown on FIG. 20.

Plasmid pMON38 is a clone of the pTiT37 HindIII-23 fragment inserted inthe HindIII site of pBR327 (Soberon, et al 1980). pMON38 DNA (20 ug) wasdigested with EcoRI (20 units, 2 hours, 37° C.) and calf alkalinephosphatase (0.2 units, 1 hour, 37° C.) The pMON38 DNA reaction wasextracted with phenol, precipitated with ethanol, dried and resuspendedin 20 ul of 10 mM Tris-HCl, 1 mM EDTA, pH 8.

0.2 ug of the cleaved pMON38 DNA was added to the chimeric gene mixturedescribed above. The mixture was treated with T4 DNA ligase (4 units, 1hour, 22° C.) and mixed with Rb chloride-treated E. coli C600 recA56cells to obtain transformation. After plating with selection forampicillin-resistant (200 ug/ml) colonies, 63 potential candidates wereobtained. Alkaline mini-preps of plasmid DNA were made from 12 of theseand screened by restriction endonuclease digestion for the properconstructs. Plasmid DNA's that contained a 1.5 kb EcoRI fragment and anew BgII site were digested with BamHI to determine the orientation ofthe 1.5 kb EcoRI fragment. One of each insert orientation was picked.One plasmid was designated pMON75 and the other pMON76, as shown in FIG.20. DNA from these plasmids were prepared as described in previousexamples.

REFERENCES

-   A. Bale et al, Mut. Res. 59: 157 (1979)-   F. Bolivar, Gene 4: 121 (1978)-   A. Braun and H. Wood, Proc. Natl. Acad. Sci. USA 73: 496 (1976)-   M. D. Chilton et al, Cell 11: 263 (1977)-   A. Colman et al, Eur. J. Biochem 91: 303-310 (1978)-   T. Currier and E. Nester, J. Bact. 126: 157 (1976)-   M. Davey et al, Plant Sci. Lett. 18: 307 (1980)-   R. W. Davis et al, Advanced Bacterial Genetics Cold Spring Harbor    Laboratory, New York, (1980)-   H. De Greve et al, Plasmid 6: 235 (1981)-   G. Ditta et al, Proc. Natl. Acad. Sci. USA 77: 7347 (1980)-   D. Garfinkel et al, Cell 27: 143 (1981)-   S. Hasezawa et al, Mol. Gen. Genet. 182: 206 (1981)-   J. Hernalsteens et al, Nature 287: 654 (1980)-   D. Ish-Horowicz and J. F. Burke, Nucleic Acids Res. 9: 2989-2998    (1981)-   B. Koekman et al, J. Bacteriol. 141: 129 (1979)-   F. Krens et al, Nature 296: 72 (1982)-   J. Leemans et al, J. Mol. Appl. Genet. 1: 149 (1981)-   J. Leemans et al, The EMBO J. 1: 147 (1982)-   A. L. Lehninger, Biochemistry, 2nd ed. (Worth Publ., 1975)-   P. Lurquin, Nucleic Acids. Res. 6: 3773 (1979)-   T. Maniatis et al, Molecular Cloning, A Laboratory Manual (Cold    Spring Harbor Labs, 1982)-   L. Marton et al, Nature 277: 129 (1979)-   T. Matzke and M-D Chilton, J. Mol. Appl. Genet. 1: 39 (1981)-   J. Messing et al, Nucleic Acids Res. 9: 309 (1981)-   J. Messing and J. Vieira, Gene 19:.269-276 (1982)-   J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor    Laboratory, N.Y. (1972)-   N. Murray et al, J. Mol. Biol. 132: 493 (1979)-   G. Ooms et al, Plasmid 7: 15 (1982)-   L. Otten and R. Schilperoort, Biochim. Biophys Acta 527: 497 (1978)-   L. Otten, Mol. Gen. Genet. 183: 209 (1981)-   R. Roberts, Nucleic Acids Res. 10: r117 (1982)-   A. Rorsch and R. Schilperoort, Genetic Engineering, 189    Elsevier/North Holland, N.Y. (1978)-   J. K. Setlow and A. Hollaender, Genetic Engineering, Principles and    Methods (Plenum Press 1979)-   X. Soberon et al, Gene 9: 287 (1980)-   L. Stryer, Biochemistry, 2nd. ed. (W. H. Freeman and Co., 1981)-   M. Thomashow et al, Cell 19: 729 (1980)-   B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci.: 615-619    (1979)-   L. Willmitzer et al, Nature 287: 359 (1980)-   L. Willmitzer et al, The EMBO J. 1: 139 (1982)-   N. Yadev et al, Nature 287: 1 (1980)-   F. Yang et al, Mol. Gen. Genet. 177: 707 (1980)-   P. Zambryski et al, J. Mol. Appl. Genet. 1:361 (1982)-   S. Adams et al, Abstract #149, 183rd Meeting of the Amer. Chemical    Society (1982)-   N. Amrhein et al, Plant Physiol. 66: 830 (1980)-   E Auerswald et al, Cold Spr. Hbr. Symp. Quant. Biol. 45: 107 (1981)-   S. Beaucage and M. Carruthers, Tetrahedron Lett. 22: 1859 (1981)-   E. Beck et al, Gene 19: 327 (1982)-   J. Beggs, Nature 275: 104 (1978)-   D. Berg et al, Proc. Natl. Acad. Sci. USA 76: 3628 (1975)-   M. Capecchi, Cell 22: 479 (1980)-   A. C. Y. Chang and S. U. Cohen, J. Bacteriol. 134: 1141-1156 (1978)-   F. Colbece-Garapin, et al, J. Mol. Biol. 150:1-14 (1981)-   Covey, S. N., G. P. Lomonosoff and R. Hull (1981) Nucleic Acids    Res., 9:6735-6747-   T. Currier and E. Nester, J. Bact. 126: 157 (1976)-   M. Davey et al, Plant Sci. Lett. 18: 307 (1980)-   Dudley, R. et al., Virology 117:19 (1982)-   R. Fischer and R. Goldberg, Cell 29: 651 (1982)-   R. Fraley and D. Papahadjopoulos, Current Topics in Microbiology and    Immunology 96: 171 (1981)-   Fraley, R. T., R. B. Horsch, A. Matzke, M. D. Chilton, W. S. Chilton    and P. R. Sanders, Plant Molecular Biology 3, 371-378 (1984)-   Frank, A., H. Guilley, G. Joward, K. Richards and L. Hirth, Cell 21,    285-294 (1980)-   Gardner, R. C. et al., Nucleic Acids Research Vol. 9, No. 12:287    (1981)-   D. Garfinkel et al, Cell 27: 143 (1981)-   L. Guarente, et al, Science 209: 1428-1430 (1980)-   Howarth, A. S. et al., Virology 112:678 (1981)-   J. Hyldig-Nielsen, Nucleic Acids Res. 10: 689 (1982)-   K. Itakura, et al, Science 198: 1056-1063 (1977)-   A. Jimenez and J. Davies, Nature 287: 869 (1980).-   M. Kozak, Cell 15: 1109 (1978)-   S. McKnight, Cell 31: 355 (1982)-   J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor    Laboratory, N.Y. (1972)-   J. Miller and W. Reznikof, The Operon, 2nd edition, Cold Spring    Harbor Laboratory, New York (1982)-   N. Murray et al, J. Mol. Biol. 132: 493 (1979)-   H. Pederson et al, Cell 29: 1015 (1982)-   A. Petit and J. Tempe, Mol. Gen. Genet. 167: 145 (1978)-   J. Pittard and B. Wallace, J. Bacteriol. 91: 1494 (1966)-   C. M. Radding, Annu. Rev. Biochem. 47: 847-880 (1978)-   N. Rao and S. Rogers, Gene 7: 79 (1979)-   M. Rassoulzadegan et al, Nature 295: 257 (1982)-   T. Roberts et al, Proc. Natl. Acad. Sci. USA 76: 760 (1979)-   K. Sakaguchi and M. Okanishi, Molecular Breeding and Genetics of    Applied Microorganisms, Kodansha/Academic Press (1981)-   D. Sciaky et al, Plasmid 1: 238 (1978)-   D. Shah et al, Proc. Natl. Acad. Sci. USA 79: 1022 (1982)-   T. Shibata et al, Proc. Natl. Acad. Sci. USA 76: 1638-1642 (1979)-   P. Southern and P. Berg, J. Mol. Appl. Gen. 1: 327-341 (1982)-   K. Struhl et al, Proc. Natl. Acad. Sci. USA 75: 1929 (1979)-   L. Stryer, Biochemistry, 2nd edition (Freeman & Co., San Francisco,    1981)-   J. Vieira and J. Messing, Gene 19: 259 (1982)-   T.-K. Wong and E. Neumann, Bioch. Biophys. Res. Comm. 107: 584    (1982)-   R. Woychik et al, Nucleic Acids Res. 10: 7197 (1982)

1. A co-integrate Ti plasmid for use in transforming plant cellscomprising a T-DNA region having the following elements, in sequence:(a) a first T-DNA border sequence; (b) a plant gene comprising a 5′promoter region from a gene which exists naturally in a plant cell ornaturally enters a plant cell, a structural coding sequence encoding apolypeptide expressable in plant cells and a 3′ non-translated regionencoding a polyadenylation signal, said 5′ promoter region and 3′non-translated region being operably linked to said structural codingsequence; and (c) a second T-DNA border sequence, wherein said T-DNAborder sequences enable the transfer and incorporation of T-DNA into thegenome of a plant cell and there are no plant tumorigenic genes betweenand including the two T-DNA border sequences which would render atransformed plant cell tumorous or incapable of regeneration into amorphologically normal plant.
 2. A plasmid of claim 1 in which the plantgene functions as a selectable marker in transformed plant cells.
 3. Aplasmid of claim 2 in which the chimeric gene causes the plant cells tobe antibiotic resistant.
 4. A plasmid of claim 3 in which the chimericgene expresses neomycin phosophotransferase.
 5. A plasmid of claim 4wherein the chimeric gene comprises a promoter derived from a plantgene.
 6. A plasmid of claim 1 which is obtained by in vivo recombinationbetween a plant tumor inducing plasmid and a chimeric plasmid comprisinga gene which functions in plants comprising in sequence: (a) a region ofhomology which is capable of causing in vivo recombination of thechimeric plasmid with a plant tumor inducing plasmid of Agrobacterium;(b) a plant gene comprising a 5′ promoter region from a gene whichexists naturally in a plant cell or naturally enters a plant cell, astructural coding sequence encoding a polypeptide which functions inplant cells as a selectable marker and a 3′ non-translated regionencoding a polyadenylation signal, said promoter and 3′ non-translatedregion being operably linked to said structural coding sequence; and (c)an Agrobacterium plasmid T-DNA border sequence which enables thetransfer and incorporation of T-DNA into the genome of a plant cell;said chimeric plasmid containing no plant tumorigenic genes between andincluding the region of homology and the T-DNA border sequence.
 7. Aplasmid of claim 6 in which the plant tumor inducing plasmid is awild-type Ti plasmid.
 8. A plasmid of claim 6 in which the plant tumorinducing plasmid is a disarmed Ti plasmid in which the tumorigenic genesand T-DNA left border has been removed.
 9. A co-integrate Ti plasmid foruse in transforming plant cells having a first gene which functions as aselectable marker gene in bacteria and a T-DNA region comprising: (a) afirst T-DNA border sequence; (b) a second gene which functions as aselectable marker gene in transformed plant cells; (c) a third genewhich is expressed in plant cells; and (d) a second T-DNA bordersequence; in which said T-DNA border sequences enable the transfer andincorporation of T-DNA into the genome of a plant cell and there are noplant tumorigenic genes between and including the two T-DNA bordersequences.
 10. The plasmid of claim 9 in which (c) functions as ascorable marker gene.
 11. The plasmid of claim 9 in which (c) is achimeric gene.
 12. The plasmid of claim 9 created by a crossover eventbetween a plant tumor inducing plasmid of Agrobacterium and a chimericplasmid comprising a gene which functions in plants comprising insequence: (a) a region of homology which is capable of causing in vivorecombination of the chimeric plasmid with a plant tumor inducingplasmid of Agrobacterium; (b) a plant gene comprising a 5′ promoterregion from a gene which exists naturally in a plant cell or naturallyenters a plant cell, a structural coding sequence encoding a polypeptidewhich functions in plant cells as a selectable marker and a 3′non-translated region encoding a polyadenylation signal, said promoterand 3′ non-translated region being operably linked to said structuralcoding sequence; and (c) an Agrobacterium plasmid T-DNA border sequencewhich enables the transfer and incorporation of T-DNA into the genome ofa plant cell; said chimeric plasmid containing no plant tumorigenicgenes between and including the region of homology and the T-DNA bordersequence.
 13. The plasmid of claim 9 in which the gene of (b) imparts toplant cells antibiotic resistance.
 14. The plasmid of claim 13 in whichthe gene of (b) expresses neomycin phosphotransferase.
 15. The plasmidof claim 9 which contains no tumorigenic genes.
 16. A microorganismwhich contains a co-integrate plasmid of claim
 1. 17. A microorganismwhich contains a co-integrate_plasmid of claim
 9. 18. A microorganismwhich contains a co-integrate plasmid of claim
 14. 19. A culture ofmicroorganisms of claim
 16. 20. A culture of microorganisms whichcontains a co-integrate Ti plasmid for use in transforming plant cellscomprising a T-DNA region having the following elements, in sequence:(a) a first T-DNA border sequence; (b) a plant gene comprising a 5′promoter region from a gene which exists naturally in a plant cell ornaturally enters a plant cell, a structural coding sequence encoding apolypeptide expressable in plant cells and a 3′ non-translated regionencoding a polyadenylation signal, said 5′ promoter region and 3′non-translated region being operably linked to said structural codingsequence; and (c) a second T-DNA border sequence, wherein said T-DNAborder sequences enable the transfer and incorporation of T-DNA into thegenome of a plant cell and there are no plant tumorigenic genes betweenand including the two T-DNA border sequences which would render atransformed plant cell tumorous or incapable of regeneration into amorphologically normal plant, and wherein the culture of microorganismsis identified by ATCC number
 39266. 21. The culture of microorganismsidentified by ATCC number 39263.